WO2008002157A1

WO2008002157A1 - Apyrases and uses thereof

Info

Publication number: WO2008002157A1
Application number: PCT/NZ2007/000111
Authority: WO
Inventors: Vickery Laurence Arcus; Chunhong Chen; Nicholas John Roberts; Richard William Scott
Original assignee: Agresearch Limited
Priority date: 2006-06-27
Filing date: 2007-05-14
Publication date: 2008-01-03

Abstract

A use for an apyrase or chimeric apyrase together with a substrate and a colour reaction mixture to indicate the presence or absence of a target carbohydrate.

Description

APYRASES AND USES THEREOF

STATEMENT OF CORRESPONDING APPLICATIONS

This application is based on the Provisional specification filed in relation to New Zealand Patent Application Number 548172 and New Zealand Patent Application Number 554261 , the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention broadly relates to apyrase genes and enzymes and uses thereof.

BACKGROUND ART

Apyrases are a group of enzymes (i.e. proteins) which belong to EC 3.6.1.5 and which catalyse the removal of phosphate from nucleotide tri- and di-phosphates, such as, for example ATP

Apyrases are currently used in pyrosequencing of single nucleotide polymorphisms and removal of ATP in a variety of molecular and biochemical assays.

The human apyrase CD39 (Accession No. S73813) is most the most studied apyrase, it is also referred to as an ecto-apyrase, ATP diphosphohydrolase (ATPDase) and ecto nucleotidase. CD39 is tethered to the outside of the cell by two membrane spanning domains (one at each terminus) with the apyrase domains and conserved cysteines located outside the cell. Conserved features of the apyrase class of proteins were recognised by Handa and Guidotti (1996), Vasconcelos et al., (1996), Zimmermann et al., (1998) and Roberts et al., (1999). They contain four well defined apyrase domains in the N-terminal half, six conserved cysteines in their C-termianl half and a fifth apyrase domain near the C-terminus.

Lectin Nucleotide Phophohydrolases (LNPs) and their cDNAs were originally isolated from a variety of legume species. These were characterised as peripherally bound membrane proteins belonging to the apyrase category of enzymes in which the apyrase activity is altered by the presence of specific carbohydrates. (Etzler et al., 1999; Roberts et al., 1999).

The ecto apyrases/ecto-ATPases are anchored to the outer cell membrane by two transmembrane spanning domains (one at each terminus) whereas the LNPs and many plant apyrases are either peripherally membrane bound or soluble. Phylogenetic analyses indicates that the LNPs appear to constitute a specialized category of apyrases unique to the legumes (Roberts et al., 1999; Cohn et al., 2001). However, since the ability to undergo symbiosis with mycorrhizal fungi is not confined to leguminous plants, Etzler and Roberts (2001 ; 2005) suggested that non leguminous Myc⁺ plants possess a related apyrase that performs a similar function to LNP in this symbiosis. Indeed, carbohydrate binding of other apyrases (such as ecto ATPases from Trypanosoma rangeli) was recently reported to show elevated apyrase activity in the presence of specific carbohydrates (Fonseca et al., 2006).

Apyrases appear to be present in all 5 Kingdoms organisms; and are involved in a wide variety of functions, it was recently demonstrated that LNP from the legume Lotus japonicus is essential for both the rhizobial- and mycorrhizal-legume symbioses (Etzler and Murphy, 2002; Etzler and Roberts, 2001 ; 2005).

A problem that currently exists in the identification of whether a specific carbohydrate of interest is present in a sample. As the present methods involving lectins for such identification all require the lectin to be labeled (e.g., biotin, antidioxygenin, fluoresence tags) or some other remote method of detecting bound lectin (e.g., fluorescently labeled lectins, immunolabeled antibodies, lectin nanoparticle coatings, surface plasmon resonance) which add considerable complexity and expense to the identification of carbohydrates (Asian et al., 2004; Fromeil et al., 2005; Grϋn et al., 2006; Manimala et al., 2005; Masarova et al., 2004; Rϋdiger and Gabius 2001).

In the present invention the inventor(s) have surprisingly found that changes in apyrase activity can be monitored to indicate the presence or absence of a specific carbohydrate. The exterior surfaces of cells are decorated with carbohydrates; the types of carbohydrate vary between different cell types. This phenomenon can be exploited to develop a new generation of tools for detecting contaminates in blood, infection, or the presence of certain cell types (e.g. cancer cells). However, only a limited range of lectins have been identified so far, thereby limiting the types of carbohydrates that can be identified. Also, detection of binding between lectins and carbohydrates is cumbersome and time consuming. The present invention enables the development of designer apyrases for new and novel applications.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given byway of example only.

DISCLOSURE OF INVENTION According to a first aspect of the present invention there is provided a use for an apyrase or chimeric apyrase together with a substrate and a colour reaction mixture to indicate the presence or absence of a target carbohydrate.

The colour reaction is the result of carbohydrate recognition by the apyrase allosterically influencing the apyrase enzyme cleaving inorganic phosphate from a nucleotide tri- or di-phosphate. The detection of free phosphate is determined colourmetrically a reaction mixture (4 parts Ammonium Molybdate Reagent [15mM Zinc acetate, 1OmM Molybdate (added to zinc acetate), pH adjusted to 5.0 with cone HCI] to 1 part Reducing Agent [prepared fresh, 10% ascorbic acid, pH adjusted to 5.0 with NaOH]) and incubated at 30⁰C for 10 minutes. Absorbance at λ = 630nm is determined using a plate reader.

According to a second aspect of the present invention there is provided a chimeric apyrase with altered carbohydrate binding compared to known native apyrases.

According to a third aspect of the present invention there is provided a chimeric apyrase gene encoding a chimeric apyrase having altered carbohydrate binding compared to known native apyrases.

According to a fourth aspect of the present invention there is provided a use of an apyrase or chimeric apyrase to remove or isolate a target carbohydrate from a liquid sample.

The target carbohydrate may be part of a larger molecule such as a protein (glycoprotein) or lipid (glycolipid), in turn the complete glycoprotein or glycolipid may be attached to a larger complex or whole cell.

One preferred way an apyrase or chimeric apyrase may be used to remove or isolate a carbohydrate is via affinity chromatography where the apyrase is immobilised on a column or solid support, e.g., via a N- or C-terminal affinity tag, covalent spacer, direct coupling to activated sepharaose, or indirect coupling via an immobilised specific antibody. The sample containing the target carbohydrate can either be batch processed, for example with the immobilised apyrase on beads where the liquid sample and beads are mixed together or passed over the column containing immobilised apyrase. The carbohydrate bound to the apyrase is then removed from the sample either by centrifugation or left on the column while the carbohydrate free eluate is recovered.

According to a fifth aspect of the present invention there is provided a use for nucleotide sequence information of at least two apyrases to construct a chimeric apyrase gene.

According to a sixth aspect of the present invention there is provided a method of using an apyrase or chimeric apyrase to specifically bind a target carbohydrate comprising the steps of:

a) contacting a liquid containing a target carbohydrate with an apyrase or chimeric apyrase which binds to said carbohydrate; and

b) removing said liquid from the apyrase or chimeric apyrase or vice versa.

According to a seventh aspect of the present invention there is provided a method of treating a disease ex-vivo using an apyrase or chimeric apyrase to specifically bind a target carbohydrate comprising the steps of:

a) contacting a liquid containing a target carbohydrate associated with a disease with an apyrase or chimeric apyrase which binds to said carbohydrate; and

b) removing said liquid from the apyrase or chimeric apyrase or vice versa.

One preferred method of treating diseases may be achieved by affinity chromatography where the apyrase is immobilised on a column or solid support, e.g., via a N- or C-terminal affinity tag, covalent spacer, direct coupling to activated sepharaose, or indirect coupling via an immobilised specific antibody. The sample containing the target carbohydrate can either be batch processed with the immobilised apyrase or passed over the column containing immobilised apyrase. The carbohydrate bound to the apyrase is then removed from the sample either by centrifugation or left on the column while the carbohydrate free eluate is recovered.

In one preferred embodiment the assay may immobilize apyrases or chimeric apyrases with known carbohydrate binding properties onto ELISA plates. Aqueous samples containing unknown carbohydrates versus samples containing known carbohydrates are incubated with the immobilised apyrases. The apyrase activity may preferably be determined colourometrically in each well. The level of the apyrase activity is indicative of the amount of specific carbohydrate present in the sample by virtue of the allosteric effect of carbohydrate binding on apyrase activity.

Alternatively the samples containing carbohydrates can be fixed to the ELiSA plates and then incubated with specific apyrases or chimeric apyrases. After washing the unbound apyrase or chimeric apyrase away the level of apyrase activity remaining can be determined colourometrically in each well. The presence of apyrase activity is indicative of the presence of the corresponding carbohydrate that the specific apyrase binds to. The level of apyrase activity is indicative of the amount of specific carbohydrate present.

According to an eighth aspect of the present invention there is provided an assay which includes an apyrase or chimeric apyrase which can specifically bind to a target carbohydrate.

According to a ninth aspect of the present invention there is provided a method for identifying the presence of a carbohydrate known to specifically bind to the carbohydrate of interest, comprising the steps of:

a) adding together apyrase or chimeric apyrase, a colour reaction mixture a substrate, and a sample including a carbohydrate and;

b) measuring the enzymatic activity of the ayprase or chimeric apyrase on the substrate.

c) comparing the measured absorbance at step b) to the known absorbance of unbound activity of the apyrase or chimeric apyrase.

One preferred way this may be achieved is by immobilising apyrases or chimeric apyrases with known carbohydrate binding properties onto ELISA plates. Aqueous samples containing unknown carbohydrates versus samples containing known carbohydrates are incubated with the immobilised apyrases. The apyrase activity is then determined colourometrically in each well. The level of the apyrase activity is indicative of the amount of specific carbohydrate present in the sample by virtue of the allosteric effect of carbohydrate binding on apyrase activity.

Alternatively the samples containing carbohydrates can be fixed to the ELISA plates and then incubated with specific apyrases or chimeric apyrases. After washing the unbound apyrase or chimeric apyrase off the level of apyrase activity can be determined by adding suitable substrate and buffer to each well and determining the level of free phosphate released colourometrically. The presence of apyrase activity is indicative of the presence of the corresponding carbohydrate that the specific apyrase binds to. The level of apyrase activity is indicative of the amount of specific carbohydrate present. Preferably, absorbance may be measured using any suitable plate reader, such as a TECAN microplate reader.

The level of apyrase or chimeric apryase activity may be determined by comparing the absorbance of free phosphate in the colour reaction mixture (cleaved from nucleotide substrates by the apyrase) with that of known quantities of free inorganic phosphate.incubated in the colour reaction mixture in the absence of apyrase, nucleotides etc.

The influence of specific carbohydrates on apyrase or chimeric apyrase activity may be determined by comparing the absorbance of free phosphate in the colour reaction mixture (cleaved from nucleotide substrates by the apyrase) in the presence of specific carbohydrates with the absorbance of free phosphate in the colour reaction mixture (cleaved from nucleotide substrates by the apyrase) in the absence of carbohydrates. The colour reaction is the result of carbohydrate recognition by the apyrase allosterically influencing the apyrase enzyme cleaving inorganic phosphate from a nucleotide tri- or diphosphate. The detection of free phosphate is determined colourmetrically a reaction mixture (4 parts Ammonium Molybdate Reagent [15mM Zinc acetate, 1OmM Molybdate (added to zinc acetate), pH adjusted to 5.0 with cone HCI] to 1 part Reducing Agent [prepared fresh, 10% ascorbic acid, pH adjusted to 5.0 with NaOH]) and incubated at 3O⁰C for 10 minutes. Absorbance at λ = 630nm is determined using a plate reader. Preferably the construct may be operatively linked to one or more regulatory sequences if required for expression of the apyrase gene or chimeric apyrase gene of interest.

According to a further aspect of the present invention there is provided a substantially isolated nucleic acid molecule selected from the group consisting of:

a) the sequence shown in SEQ ID No. 1 ;

b) a complement of the sequence shown in SEQ ID No. 1 ;

c) a reverse complement to the sequences recited in a), and b); and

d) a reverse sequence to a sequence recited in c); and

e) functionally active fragments and variants of the sequences recited in a), b), c) and d).

a) the sequence shown in SEQ ID No. 3;

b) a complement of the sequence shown in SEQ ID No. 3;

c) a reverse complement to the sequences recited in a), and b); and

d) a reverse sequence to a sequence recited in c); and

a) the sequence shown in SEQ ID No. 5;

b) a complement of the sequence shown in SEQ ID No. 5; c) a reverse complement to the sequences recited in a), and b); and

d) a reverse sequence to a sequence recited in c); and

According to a further aspect of the present invention there is provided a substantially isolated apyrase which has an amino acid sequence selected from the group consisting of:

a) SEQ ID No. 2; or

b) a functionally active fragment or variant of a) sequence recited in a).

A substantially isolated apyrase which has an amino acid sequence selected from the group consisting of:

a) SEQ ID No. 4; or

b) a functionally active fragment or variant of a) sequence recited in a).

a) SEQ ID No. 6; or

b) a functionally active fragment or variant of a) sequence recited in a).

According to another aspect of the present invention there is provided an apyrase which has been substantially encoded by a nucleic acid molecule having a nucleotide sequence substantially as set forth in the group consisting of:

a) SEQ ID Nos. 1 , 3, and 5; or

b) a complement of the sequence shown in a);

c) a reverse complement to the sequences recited in a), and b); and d) a reverse sequence to a sequence recited in c); and

According to another aspect of the present invention there is provided an antibody which specifically binds to an apyrase having an amino acid sequence selected from the group consisting of:

a) SEQ ID Nos. 2, 4 or 6; or

b) a functionally active fragment or variant of a sequence recited in a).

According to another aspect of the present invention there is provided the use of nucleotide sequence information of at least two apyrases to construct a chimeric apyrase gene having altered apyrase nucleotide substrate specificity, and/or apyrase activity and/or pH optimum with respect to one or more of the native apyrases from which the nucleotide sequence information was obtained.

Preferably, the use of nucleotide sequence information may include constructing an apyrase gene having altered phosphohydrolase activity.

In some preferred embodiments the altered phosphohydrolase activity narrows the nucleotide triphosphates on which the chimeric apyrase can act.

In some furhter preferred embodiments the altered phosphohydrolase activity broadens the nucleotide tri-phosphates on which the chimeric apyrase can act.

Briefly, the above uses of sequence information involve generating chimeric apyrases by making in frame chimeric substitutions of the apyrase conserved regions (4 N-terminal and 1 C-terminal) and/or other conserved regions from different apyrases.

According to another aspect of the present invention there is provided the use of nucleotide sequence information of at least two apyrases to construct a chimeric apyrase gene having altered oligomeric status with respect to one or more of the native apyrases from which the nucleotide sequence information was obtained. Briefly, this involves generating chimeric apyrases by making in frame chimeric substitutions of the apyrase conserved regions (4 N-terminal and 1 C-terminal) and/or other conserved regions from different apyrases.

The term 'oligometric status' refers to the structural characteristics of an apyrase or chimeric apyrase and whether it is a monomer or polymer of generally between 2-4 monomeric subunits.

According to another aspect of the present invention there is provided the use of nucleotide sequence information of at least two apyrases to construct a chimeric apyrase gene having altered carbohydrate specificity with respect to one or more of the native apyrases from which the nucleotide sequence information was obtained.

According to a further aspect of the present invention there is provided a chimeric apyrase comprising a first fragment obtained from a first apyrase gene sequence and a second fragment obtained from a second apyrase gene sequence wherein said first and second apyrase gene sequences are either homologous, heterologous or isologous

Briefly, the above uses of sequence information involve generating chimeric apyrases by making in frame chimeric substitutions of apyrase conserved regions (4 N-terminal and 1 C-terminal) and/or other conserved regions from different apyrases.

According to a further aspect of the present invention there is provided a host cell which comprises one or more nucleic acid molecules substantially as described herein.

According to another aspect of the present invention there is provided a transformed cell which includes a nucleic acid molecule selected from the group consisting of:

a) SEQ ID No. 1, 3, or 5; or

b) a complement of the sequence shown in a);

According to another aspect of the present invention there is provided a host cell which includes a nucleic acid molecule selected from the group consisting of:

a) SEQ ID No. 1 , 3, or 5; or

b) a complement of the sequence shown in a);

c) a reverse complement to the sequences recited in a), and b); and

d) a reverse sequence to a sequence recited in c); and

According to another aspect of the present invention there is provided the use of a nucleic acid molecule selected from the group consisting of:

a) SEQ ID No. 1, 3, or 5; or

b) a complement of the sequence shown in a);

c) a reverse complement to the sequences recited in a), and b); and

d) a reverse sequence to a sequence recited in c); and

as a probe.

According to a further aspect of the present invention there is provided a chimeric apyrase comprising an N-terminus derived from a first apyrase gene sequence and a C-terminus from a second apyrase gene sequence, wherein said first and second apyrase gene sequences are either homologous, heterologous or isologous. According to yet a further aspect of the present invention there is provided a chimeric apyrase substantially as described herein with reference to any example and/or drawing thereof.

According to a further aspect of the present invention there is provided the use of a nucleic acid molecule of the present invention substantially as described above to identify apyrases from different organisms.

According to a further aspect there is provided an oligonucleotide comprising at least 14-20 contiguous nucleotides selected from a nucleic acid molecule of the present invention.

According to a further aspect there is provided the use of an oligonucleotide of the present invention to identify or isolate an apyrase gene.

According to a further aspect of the present invention there is provided a host cell which includes a chimeric apyrase gene.

According to a further aspect of the present invention there is provided a transformed cell which includes a chimeric apyrase gene.

According to a further aspect of the present invention there is provided a chimeric apyrase gene encoding a chimeric apyrase substantially as described above.

The present invention broadly relates to ayprases (i.e. apyrase enzymes refer EC 3.6.1.5) and genes encoding apyrases. The present invention also relates to both peripherally bound and soluble apyrases such as found in plants, animals, fungi, protozoa and bacteria.

The present invention is equally applicable for those integral membrane bound apyrases that have been generated as soluble recombinant apyrases by removal of their membrane spanning/anchoring domain(s), e.g., CD39.

The term "native apyrase" as used herein refers to an apyrase as found in nature. The term "apyrase" as used herein refers to a family of enzymes (i.e. proteins belonging to class EC 3.6.1.5) which has phosphohydrolase activity (i.e. catalyses the removal of phosphate from ATP and ADP and other nucleotide tri-phosphates and di-phosphates including UTP and UDP) and which can also bind to a specific carbohydrate compound.

The term "chimera" and "chimeric" as used herein relates to:

a) a recombinant apyrase gene substantially formed by two or more heterologous apyrase nucleic acid sequences; or

b) an apyrase encoded by two or more heterologous apyrase nucleic acid sequences joined to form a recombinant apryase gene; or

c) an apyrase having two or more heterologous domains.

Nucleic acid molecules according to the invention may be full-length genes or part thereof, and may also be referred to as "nucleic acids", "nucleic acid fragments" and "nucleotide sequences" in this specification.

The nucleic acid fragment may be of any suitable type and includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) that is single- or double-stranded, optionally containing synthetic, non- natural or altered nucleotide bases, and combinations thereof.

The term "variant" as used herein refers to nucleotide and polypeptide sequences wherein the nucleotide or amino acid sequence exhibits a minimum homology selected from a range consisting of: 60-69%; 70-74%; 75-79%; 80-84%; 85-89%; 90-94%; or 95-99%, in relation to the nucleotide and amino acid sequences of the present invention as shown in the sequence listing or as otherwise represented.

Preferably, the term "variant" as used herein refers to nucleotide and polypeptide sequences wherein the nucleotide or amino acid sequence exhibits a minimum homology selected from a percentage integer falling within a group of ranges consisting of: 60-69%; 70-79%; 80-89%; or 90-99%; in relation to the nucleotide and amino acid sequences of the present invention as shown in the sequence listing or as otherwise represented.

A fragment of a nucleic acid is a portion of the nucleic acid that is less than full length and comprises at least a minimum sequence capable of hybridising specifically with a nucleic acid molecule according to the present invention (or a sequence complementary thereto) under stringent conditions as defined below. A fragment of a polypeptide is a portion of the polypeptide that is less than full length.

By "functionally active" in respect of a nucleotide sequence is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of phosphohydrolase activity and carbohydrate binding. Such variants include naturally occurring allelic variants and non-naturally occurring variants an encompasses 'conservative substitutions' wherein alteration of the nucleotide sequence results in substantially alteration of a functionally similar amino acid residue - see Creighton 1984. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Such functionally active variants and fragments include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably, the fragment has a size of at least 30 nucleotides, more preferably at least 45 nucleotides, most preferably at least 60 nucleotides.

By "functionally active" in the context of a polypeptide is meant that the fragment or variant has phosphohydrolase activity and carbohydrate binding. Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably, the fragment has a size of at least 10 amino acids, more preferably at least 15 amino acids, most preferably at least 20 amino acids.

The term "gene" as used herein refers to a nucleic acid molecule comprising an ordered series of nucleotides that encodes a gene product (i.e. specific protein) such as an apyrase. The term "construct" as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest. In general a construct may include the gene or genes of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto.

The term "operably linked" or grammatical variant thereof as used herein means that the regulatory sequences necessary for expression of the gene of interest are placed in the nucleic acid molecule in the appropriate positions relative to the gene to enable expression of the gene.

As used herein the term "regulatory sequences" refers to certain nucleic acid sequences such as origins of replication, promoters, enhancers, polyadenylation signals, terminators and the like, that enable expression of the nucleic acid molecule of interest.

Preferably, said regulatory element is upstream of said nucleic acid and said terminator is downstream of said nucleic acid.

The term "vector" as used herein encompasses both cloning and expression vectors. Vectors are often recombinant molecules containing nucleic acid molecules from several sources.

A "cloning vector" refers to a nucleic acid molecule originating or derived from a virus, a plasmid or a cell of a higher organism into which another exogenous (foreign) nucleic acid molecule of interest, of appropriate size can be integrated without loss of the vector's capacity for self-replication. Thus vectors can be used to introduce at least one foreign nucleic acid molecule of interest (e.g. gene of interest) into host cells, where the gene can be reproduced in large quantities.

An "expression vector" refers to a cloning vector which also contains the necessary regulatory sequences to allow for transcription and translation of the integrated gene of interest, so that the gene product of the gene can be expressed. In a preferred embodiment of this aspect of the invention, the vector may include at least one regulatory element, such as a promoter, a nucleic acid according to the present invention as a terminator; said regulatory element, nucleic acid and terminator being operatively linked.

The vector may be of any suitable type and may be viral or non-viral.

The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector.

As used herein, a "transformed cell" is a cell into which (or into an ancestor of which) there has been introduced, by means of recombinant DNA techniques, a nucleic acid molecule of interest. The nucleic acid of interest will typically encode a peptide or protein. The transformed cell may express the sequence of interest or may be used only to propagate the sequence. The term "transformed" may be used herein to embrace any method of introducing exogenous nucleic acids including, but not limited to, transformation, transfection, electroporation, microinjection, viral-mediated transfection, and the like.

The term "exogenous" as used herein, refers to matter such as DNA originating outside an organism.

The term 'heterologous' as used herein refers to a gene which has been obtained from an organism of a different species.

The term 'homologous' as used herein refers to a gene which has been obtained from an organism which is the same species but of a different genotype.

The term 'isologous' as used herein refers to a gene which has been obtained from a different organism of the same genotype, such as an identical twin or a clone.

By "an effective amount" is meant an amount sufficient to result in an identifiable phenotypic trait. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of application, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Sambrook et al, (1989), the entire disclosure of which is incorporated herein by reference.

It should be appreciated that the term "antibody" encompasses fragments or analogues of antibodies which retain the ability to bind to a polypeptide of the invention, including but not limited to Fr, F(ab)₂ fragments, ScFv molecules and the like. The antibody may be polyclonal but is preferably monoclonal.

It will also be understood that the term "comprises" (or its grammatical variants) as used in this specification is equivalent to the term "includes" and should not be taken as excluding the presence of other elements or features.

The terms "complement," "reverse complement," and "reverse sequence," as used herein are best illustrated by the following example. For the sequence 5' AGGACC 3', the complement, reverse complement, and reverse sequence are as follows:

Complement 3' TCCTGG 5'

Reverse Complement 3' GGTCCT 5'

Reverse Sequence 5' CCAGGA 3'

The term "protein (or polypeptide or peptide)" refers to a protein encoded by a nucleic acid molecule of the invention, including fragments, mutations and homologs or analogs having phosphohydrolase and carbohydrate binding activity. The protein or polypeptide or peptide of the invention can be isolated from a natural source, produced by the expression of a recombinant nucleic acid molecule, or can be chemically synthesized.

It is to be clearly understood that the invention also encompasses peptide analogues, which include but are not limited to the following:

1. Compounds in which one or more amino acids are replaced by its corresponding D- amino acid. The skilled person will be aware that retro-inverso amino acid sequences can be synthesised by standard methods; see for example Choreo and Goodman, 1993;

2. Peptidomimetic compounds, in which the peptide bond is replaced by a structure more resistant to metabolic degradation. See for example Olson et al, 1993; and

3. Compounds in which individual amino acids are replaced by analogous structures for example, gem-diaminoalkyl groups or alkylmalonyl groups, with or without modified termini or alkyl, acyl or amine substitutions to modify their charge.

The use of such alternative structures can provide significantly longer half-life in the body, since they are more resistant to breakdown under physiological conditions.

Methods for combinatorial synthesis of peptide analogues and for screening of peptides and peptide analogues are well known in the art (see for example Gallop et al, 1994; Hogan, 1997).

For the purposes of this specification, the term "peptide and peptide analogue" includes compounds made up of units which have an amino and carboxy terminus separated in a 1,2, 1,3, 1,4 or larger substitution pattern. This includes the 20 naturally-occurring or "common" α-amino acids, in either the L or D configuration, the biosynthetically-available or "uncommon" amino acids not usually found in proteins, such as 4-hydroxyproIine, 5-hydroxylysine, citrulline and ornithine; synthetically-derived α- amino acids, such as α-methylalanine, norleucine, norvaline, Ca- and Λ/-alkylated amino acids, homocysteine, and homoserine; and many others as known in the art.

It also includes compounds that have an amine and carboxyl functional group separated in a 1 ,3 or larger substitution pattern, such as β-alanine, γ-amino butyric acid, Freidinger lactam (Freidinger et al,

1982), the bicyclic dipeptide (BTD) (Freidinger et al, 1982; Nagai and Sato, 1985), amino-methyl benzoic acid (Smythe and von Itzstein, 1994), and others well known in the art. Statine-like isosteres, hydroxyethylene isosteres, reduced amide bond isosteres, thioamide isosteres, urea isosteres, carbamate isosteres, thioether isosteres, vinyl isosteres and other amide bond isosteres known to the art are also useful for the purposes of the invention. A "common" amino acid is a L-amino acid selected from the group consisting of glycine, leucine, isoleucine, valine, alanine, phenylalanine, tyrosine, tryptophan, aspartate, asparagine, glutamate, glutamine, cysteine, methionine, arginine, lysine, proline, serine, threonine and histidine. These are referred to herein by their conventional three-letter or one-letter abbreviations.

An "uncommon" amino-acid includes, but is not restricted to, one selected from the group consisting of D-amino acids, homo-amino acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids (other than phenylalanine, tyrosine and tryptophan), ortho-, meta- or para-am inobenzoic acid, ornithine, citrulline, norleucine, α-glutamic acid, aminobutyric acid (Abu), and α-α disubstituted amino acids.

As used herein the term "primers" refers to short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length, which are annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, preferably a DNA polymerase. Primer pairs can be used for amplification of a nucleic acid sequence, e.g. by the polymerase chain reaction (PCR) or other nucleic acid amplification methods well known in the art. PCR-primer pairs can be derived from the sequence of a nucleic acid according to the present invention, for example, by using computer programs intended for that purpose such as Primer (Version 0.5© 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).

As used herein the term "probes" refer single-stranded oligonucleotides with a known nucleotide sequence which is labelled in some way (for example, radioactively, fluorescently or immunologically), which are used to find and mark a target DNA or RNA sequence by hybridizing to it.

The term 'oligonucleotide' as used herein refers to a short singled stranded nucleic acid molecule which can hybridise to a complementary portion of an apyrase gene under stringent conditions.

The nucleic acid fragments of the present invention may be used to isolate cDNAs and genes encoding homologous proteins from the same organism or other species. Additionally, genes encoding other apyrase enzymes, either as cDNAs or genomic DNAs, may be isolated directly by using all or a portion of the nucleic acid fragments of the present invention as hybridisation probes to screen libraries from the desired organism employing the methodology known to those skilled in the art. Specific oligonucleotide probes based upon the nucleic acid sequences of the present invention can be designed and synthesized by methods known in the art. Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labelling, nick translation, or end-labelling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the sequences of the present invention. The resulting amplification products can be labelled directly during amplification reactions or labelled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

In addition, two short segments of the nucleic acid fragments of the present invention may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the nucleic acid fragments of the present invention, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding apyrase genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, those skilled in the art can follow the RACE protocol (Frohman et al. (1988) Proc, Natl. Acad Sci. USA 85:8998, the entire disclosure of which is incorporated herein by reference) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Using commercially available 3' RACE and 5' RACE systems (BRL), specific 3' or 5' cDNA fragments can be isolated (Ohara et al. 1989; Loh et al. 1989). Products generated by the 3' and 5' RACE procedures can be combined to generate full-length cDNAs. In a further embodiment of this aspect of the invention, there is provided a polypeptide recombinantly produced from a nucleic acid according to the present invention. Techniques for recombinantly producing polypeptides are known to those skilled in the art.

Thus, preferred embodiments of the present invention have a number of advantages over prior art carbohydrate identification and isolation techniques, as well as providing new uses for apyrases and new apyrases which can be used in a variety of different applications in relation to carbohydrates.

BRIEF DESCRIPTION OF DRAWINGS

Further aspects of the present invention will become apparent from the following description which is given byway of example only and with reference to the accompanying drawings in which:

Figure 1A. Nucleic acid sequence of 4WC cDNA (open reading frame indicated by overhead bar) and translated sequence of 4WC open reading frame.

Figure 1B. Nucleic acid sequence of 7WC cDNA (open reading frame indicated by overhead bar) and translated sequence of 7WC open reading frame.

Figure 1C. Nucleic acid sequence of 6RG cDNA (open reading frame indicated by overhead bar) and translated sequence of 6RG open reading frame.

Figure 2. Dendrogram analysis of aligned translated apyrase open reading frames and corresponding signal sequence prediction. Grey highlight indicates Lolium perenne clone 6RG, Trifolium repens clones 7WC and 4WC, Dolichos biflorus clone Db-LNP (AF139807) and Homo sapiens clone CD39 (S73813).

Figure 3A. Vector map of C-terminal 6RG::His in pET28.

Figure 3B. Sequence feature map of C-terminal 6RG::His in pET28. Nucleotide sequence encoding mature peptide plust His tag is shown in grey highlight. Figure 4. Coomassiee stained SDS-PAGE analysis of recombinant C-terminal 6RG in pET28 expressed in E. coli. Arrows indicate recombinant 6RG.

Figure 5A. Vector map of mature 4WC::His in pET28.

Figure 5B. Sequence feature map of mature 4WC::His in pET28. Nucleotide sequence encoding mature peptide plust His tag is shown in grey highlight

Figure 6A. Vector map of mature 7WC::His in pET28.

Figure 6B. Sequence feature map of mature 7WC::His in pET28. Nucleotide sequence encoding mature peptide plust His tag is shown in grey highlight.

Figure 7. Coomassie stained SDS-PAGE analysis of recombinant mature 4WC in pET28 expressed in E. coli. Arrows indicate recombinant 4WC.

Figure 8. Coomassie stained SDS-PAGE analysis of recombinant mature 7WC in pET28 expressed in E. coli. Arrows indicate recombinant 7WC.

Figure 9. lmmunoblot analysis to determine antisera titre of anti-6RG.

A) Anti-6RG antibody screened against purified recombninant mature 6RG and C-terminal 6RG. Anti-6RGcc antibodies were shown to recognise both mature 6RG (expected size = 47kD) and C- terminal 6RG (expected size = 26kD). The relative sizes of the multiple bands detected on the lmmunoblot containing 6RGcc indicated that there were multimers of the C-terminal 6RG peptide.

B) 100ng of C-terminal 6RG peptide was run on a urea / SDS-PAGE denaturing, reducing gel, blotted and again screened with anti-6RG antibodies. Only a single peptide band was detected (down to a titre of 1 :25,600).

Figure 10. SDS-PAGE/immunoblot analysis to determine anti-6RG sensitivity to recombinant mature 6RG. Figure 11. SDS-PAGE/immunoblot analysis to determine antisera titre of anti-4WC. Purified antigen (protein 4WC) was run on a 10% SDS PAGE as a preparative gel then was transferred to PVDF membrane that was cut to strips. Every strip was tested a different titre. Blotting was detected by the chemiluminescence method.

Figure 12. SDS-PAGE/immunoblot analysis to determine anti-4WC sensitivity to 4WC. A range of purified antigen (100ng, 50ng 25ng, 12.5ng and 6.25ng) was run on a 10% SDS PAGE and then transferred to PVDF membrane. Two concentrations of antibody titres (1 :1000 and 1 :500) were tested against the membranes and the blotting was detected by the chemiluminescence method.

Figure 13. SDS-PAGE/immunoblot analysis to determine anti 4WC specificity. Purified proteins 6rg and 7wc (250ng) were run on SDS PAGE and transferred to PVDF membrane. High titres of antibody 4wc (1 :100 and 1 :200) were used; the blot was detected by the chemiluminescence method.

Figure 14. SDS-PAGE/immunoblot analysis to determine antisera titre of anti-7WC. Purified antigen (protein 7wc) was run a 10% SDS PAGE as a preparative gel then was transferred to PVDF membrane that was cut into strips. Every strip was tested a different titre. Blotting was detected by the chemiluminescence method.

Figure 15. SDS-PAGE/immunoblot analysis to determine anti-7WC sensitivity to 7WC. A range of purified antigen (100ng, 50ng 25ng, 12.5ng and 6.25ng) was run a 10% SDS PAGE and then transferred to PVDF membrane. Two concentrations of antibody titres (1 :1000 and 1:500) were against the membranes and the blotting was detected by the chemiluminescence method.

Figure 16. SDS-PAGE/immunoblot analysis to determine anti-7WC specificity. Determination of Anti 7WC cross reactivity to recombinant 4WC and Lotus japonics root extract by immunoblot. Antibody titre = 1 :400. 80μl root extract = 21 mg (FW) equivalents of Lotus roots. Figure 17. Schematic diagram of two rounds of PCR carried out to generate 7WC, 4WC and 6RG with a 3' Thrombin-His tag

Figure 18. Results of Pfu amplification of 7WC for cloning into pENf R-D, and 7WC-T and 4WC-T with an introduced thrombin cleavage site to be used as template in a second round of PCR. Figure 19. Results of second Pfu amplification generating 7WC & 4WC with a 3' Thrombin-His tag, for cloning into pENTR-D.

Figure 20. Results of Triple master and Taq amplification of 4WC and 6RG, respectively, for cloning into pENTR-D

Figure 21. Results of second Taq amplification generating 6RG with a 3' Thrombin-His tag, for cloning into pENTR-D.

Figure 22. Plasmid maps for 7WC with and without Thrombin His tag in the pENTR-D vector (pMW2 & pMW1 , respectively).

Figure 23. Plasmid map for 4WC + Thrombin His tag in the pENTR-D vector (pMW3).

Figure 24. Plasmid maps for 6RG with and without Thrombin-His tag in the pENTR-D vector (pENTRD-6RG & pCH32, respectively).

Figure 25. Plasmid map for 4WC in the pENTR-D vector (pCH33).

Figure 26. Results of restriction enzyme digests of putative pCH32 & 33 plasmids.

Figure 27. Results of restriction enzyme digests of putative pENTRD-6RG plasmids

Figure 28. Results of restriction enzyme digests to linearise pMW1 , pMW2 and pMW3 plasmids. Figure 29. SDS-PAGE/coomassie (A) and immunoblot (B) analysis of fractions from Ni2+ column affinity purification of insect cell/baculovirus expressed 6xHis tagged 6RG.

Figure 3OA. SDS-PAGE analysis of elutes of 4WC:His from cell pellets - Silver staining Figure 3OB. SDS-PAGE analysis of elutes of 4WC:His from cell pellets - Western blot

Figure 31A. SDS-PAGE analysis of elutes of 4WC:His from medium - Silver staining

Figure 31 B. SDS-PAGE analysis of elutes of 4WC:His from medium - Western blot

-Figure 32. SDS-PAGE analysis of elutes of 7Wc and 7WC:His from cell pellets and media. ^•?

Figure 33. Sephacryl S-100HR size exclusion analysis of 6RG. Top left - SDS-PAGE/coomassie gel analysis of size exclusion fractions. Top right - Size exclusion detector results (arrow indicating the peak eluting at >100KDa corresponds to the void volume of the column, further analysis of this fraction revealed that it contains no protein; the last elution peak (31 kDa) is inactive further analysis of this fraction revealed that it contains no protein). Bottom - SDS-PAGE/silver stain analysis of active apyrase fractions collected from the size exclusion column.

Figure 34. DS-PAGE/silver stained gel of phases sampled during mucin column purification of media-derived 4WC at a range of pH values. Lane headings indicate relevant pH and purification phases; CE = crude extract, FT = flow through, LW = last wash, E = eluate. White arrow indicates position of band for 4WC positive control sample.

Figure 35. SDS-PAGE/immunoblot analysis of phases sampled during mucin column purification of media-derived 4WC at a range of pH values.

Figure 36. Silver stained SDS-PAGE gel of phases sampled during mucin column purification of cell lysate-derived 4WC at a range of pH values. Lane headings indicate relevant pH and purification phases; CE = crude extract, FT = flow through, LW = last wash, E = eluate.

Figure 37. lmmunoblot analysis of phases sampled during mucin column purification of cell lysate-derived 4WC at a range of pH values

Figure 38. Top - SDS-PAGE analysis of 4WC protein purity from medium using a mucin colum. Left - Silver stain. Right - Immnoblot. Bottom - SDS-PAGE/silver stain analysis of the recombinant 4WC from medium after ion exchange. Lanes 2, 3 and 4 are the 4WC before loading onto the ion exchange column. Load 5 and 6 are two eluted fractions which showed a higher absorbance at A280.

Figure 39A. SDS-PAGE analysis of protein 4WC from cell pellets after Mucin column purification - Silver staining.

Figure 39B. SDS-PAGE analysis of protein 4WC from cell pellets after Mucin column purification - lmmno blot +ve Ct: 4WC from E. coli, CE: crude extract. FT: flow through, LW: last wash.

Figure 40. Effect of pH on 4WC apyrase activity using 5mM ADP as substrate.

Figure 41. Effect of pH on 4WC::His apyrase activity using 5mM ADP as substrate.

Figure 42. Determination of substrate preference for 4WC.

Figure 43. Comparison of ATP and ADP substrate preference for 4WC::His.

Figure 44. ATP and ADP substrate competition assay with 4WC. ATP or ADP were added in increasing amounts in the absence of any other substrate. ADP-ATPv, ADP was added at 5mM while the level of ATP was added in increasing amounts. ATP-ADPv, ATP was added at 5mM while the level of ADP was added in increasing amounts.

Figure 45. Chevillard analysis of the influence of substrate competition on reaction velocity of 4WC. At any point of common velocity (e.g., 4.5 which occurrs with either 2.5mM ATP or 1.OmM ADP) the ratio of the two substrates is considered to be 1 :1. The influence of substrate ratio on total velocity is then determined by varying the substrate concentrations over a range of ratios (p) 0:1 to 1 :0 where 0:1 = 2.5mM ATP and OmM ADP and 1 :0 = 0 mM ATP and 1 mM ADP. Under these conditions the maximum concentration of ATP (2.5mM) in the absence of ADP would result in a reaction velocity of 4.5; similarly for the maximum concentration of ADP (1mM) in the absence of ATP. When all conditions were tested we found that the activity was virtually independent of the ratio of ATP/ADP.

Figure 46. Effect of pH on 6RG:His apyrase activity. Figure 47. Determination of substrate preference for 6RG:His.

Figure 48. ATP and ADP substrate competition assay with 6RG:His. ATP or ADP were added in increasing amounts in the absence of any other substrate. ADP-ATPv, ADP was added at 5mM while the level of ATP was added in increasing amounts. ATP-ADPv, ATP was added at 5mM while the level of ADP was added in increasing amounts.

Figure 49. Effect of pH on 7WC:His apyrase activity.

Figure 50. Determination of substrate preference of 7WC:His. Assay condition: 5OmM Tris-HCI pHδ.O, 3mM CaCI2, 5mM NDPs or NTPs.

Figure 51. ATP and ADP substrate competition assay with 7WC:His. ATP or ADP were added in increasing amounts in the absence of any other substrate. ADP-ATPv, ADP was added at 5mM while the level of ATP was added in increasing amounts. ATP-ADPv, ATP was added at 5mM while the level of ADP was added in increasing amounts

Figure 52. Chevillard analysis of the influence of substrate competition on reaction velocity of 7WC:His. At any point of common velocity (e.g., 42.5 which occurrs with either 1 mM ATP or 5mM ADP) the ratio of the two substrates is considered to be 1 :1. The influence of substrate ratio on total velocity is then determined by varying the substrate concentrations over a range of ratios (p) 0:1 to 1:0 where 0:1 = 1mM ATP and OmM ADP and 1 :0 = 0 mM ATP and 5mM ADP. Under these conditions the maximum concentration of ATP (1mM) in the absence of ADP would result in a reaction velocity of 42.5; similarly for the maximum concentration of ADP (5mM) in the absence of ATP. All conditions containing a mixture of ATP and ADP gave reaction velocities greater than expected.

Figure 53. SDS-PAGE/immunoblot analysis of mucin-binding properties of 7WC:His from baculovirus+ve Ct: 7WC from E. coli, CE: crude extract. FT: flow through, LW: last wash

Figure 54. SDS-PAGE/immunoblot analysis of mucin-binding properties of 7WC:His from E. coli +ve Ct: 7WC from E. coli, CE: crude extract. FT: flow through, LW: last wash Figure 55. SDS-PAGE/immunoblot analysis of 6RG:His mucin-binding profile. +ve: 4WC from B. coli, CE: crude extract, FT: flow through, LW: last wash

Figure 56. Align X alignment of selected apyrases. Grey highlight represent conserved cysteines;

Bold face P represent semi conserved prolines, Grey blocks represent! apyrase domaiitis. Bold face, italics N represent potential N-linked glycosylation site, black horizontal bars indicate conserved plant and animal disulfide bonds, dashed horizontal bar indicates conserved animal disulfide bond.

Figure 57. Align X output of apyrase domains and C-termini from 7WC, 4WC, DbLNP, 6RG and truncated CD39. Qrp% JhighjϊgM represent conserved cysteines. Grey blocks represent apyr&se domains. Bold capital P represents semi conserved prolines. Solid horizontal black bars represent conserved plant and animal disulfide bonds. Dashed horizontal bars represent conserved animal disulfide bond

Figure 58. NNPREDICT secondary structure prediction for 7WC, 4WC, Db-LNP, 6RG and CD39. Θreyjitshitglϊt represent conserved cysteines. Bold P represent semi conserved prolines. Bold, italics N represents potential N-glycosylation site. Solid horizontal black bars represent conserved plant and animal disulfide bonds. Dashed horizontal bars represent conserved animal disulfide bond. Clear Boxes boxes represent conserved predicted beta sheet structures. Vertical cross hatched boxes represent conserved predicted alpha helices structures.

Figure 59. ExPASy ProtScale Hydrophobicity Plots of the C-terminal half of 7WC, 4WC, DbLNP, 6RG and CD39. Tall vertical grey bars indicate conserved cysteine residues, Short vertical black bars indicate semi conserved proline residues, Horizontal grey blocks indicate the positions of inter cysteine loops.

Figure 60. Nucleotide sequence and translation around the Bam HI site used co create pCH37, pCH38 and pCH43 from 4WC and 6RG.

Figure 61. Schematic diagram showing 6RG and 4WC contributions to the chimeras pCH37, pCH43, 6RG and pCH38. Figure 62. Aligned full length peptide sequences of pCH37, pCH43, 6RG, pCH38 and 4WC.

Figure 63. SDS-PAGE immunoblot and Coomassie blue time course analysis of pCH37 expression in insect cells, lmmuno blots (left) were probed with anti 4WC. Coomassie blue stained gels are shown on the right. Starting MOI values are shown on the far left.

Figure 64A. SDS-PAGE immunoblot and Coomassie stain analysis of semi purified pCH38 from both media and pellet localised protein - Immunoblot probed with anti 6RG.

Figure 64B. SDS-PAGE immunoblot and Coomassie stain analysis of semi purified pCH38 from both media and pellet localised protein - Coomassie stained gel after immunoblot transfer, red arrows indicate recombinant pCH38. Figure 65. SDS-PAGE/coomassie stain (left) and immunoblot analysis (right) of semi purified pCH43 (6RG::4WC::His). Recombinant protein was detected only in the cell pellet and not in the medium.

Figure 66. SDS-PAGE immunoblot analysis of mucin binding properties of pCH38 purified from the media (Al & All) and cell pellet (B).

AIL) lmmunoblots of column eluate at different pH values, probed with anti 6RG antiserum; All) Immunoblot of pCH38 mucin binding properties at pH 7.5, probed with anti 6RG antiserum. B SDS-PAGE immunoblot analysis of mucin binding properties of pCH38 purified from the cell pellet, lmmunoblots of column eluate and column pellets at different pH, probed with anti 6RG antiserum

Figure 67. Apyrase activity, pH optimum and substrate specificity of 4WC, 4WC::His (pMW3), 4WC::6RG::His (pCH38), 6RG::His, 6RG::4WC::His (pCH43).

Figure 68. SDS PAGE immunoblot analysis of deglycosylated denatured 4WC from the media. Deglycosylation appeared to be complete in approximately 2.5 minutes. Non-deglycosylated protein is indicated by a white arrow. Fully deglycosylated protein is indicated by a black arrow.

Figure 69. SDS PAGE immunoblot analysis of deglycosylated native 4WC from the media. Deglycosylation appeared to be complete in approximately 2 minutes. No intermediate bands were seen between the fully deglycosylated (black arrow) and non deglycosylated protein (white arrow). Figure 7OA. SDS PAGE immunoblot analysis of deglycosylated denatured 6RG.

Figure 7OB SDS PAGE silver stain analysis of deglycosylated denatured 6RG. Deglycosylation appeared to be complete in approximately 2-5 minutes. Non-deglycosylated protein is indicated by a white arrow. Fully deglycosylated protein is indicated by a black arrow.

Figure 71A. SDS PAGE immunoblot analysis of deglycosylated native 6RG.

Figure 71 B SDS PAGE silver stain analysis of deglycosylated native 6RG. Deglycosylation appeared to be complete in approximately 10 minutes. The presence of discrete bands (dashed white arrows) between the fully deglycosylated (black arrow) and non deglycosylated protein (solid white arrow) indicates that multiple potential glycosylation sites are glycosylated.

Figure 72. Schematic diagram showing the position of potential N-linked glycosylation sites relative to the apyrase domains and conserved cysteines for 7 WC, 6RG, 4WC, pCH38, pCH43, pCH45, pCH47 and pCH48.

Figure 73. Time course of recombinant 4WC expression. 4WC = wild type; pCH45 = N313- A313; pCH47 = N85-A85; pCH48 = N85-A85; N313-A313.

Figure 74. SDS-PAGE/immunoblot gel shift analysis of pCH45, pCH47 and pCH48. Black arrow indicates larger isoform seen in 4WC, pHC47 and pMW3(4WC::His), white arrow indicates larger isoform seen in pCH45 and pCH48. The lager isoform in pCH45 and pCH48 has a faster electrophoretic mobility compared to wild type and pCH47.

Figure 75. Apyrase activity of 4WC glycosylation mutants, determined from whole cell extracts. All samples are normalised against the apyrase activity found in untransformed Sf9 cells. 4WC = wild type recombinant protein ; pCH45 (N313-A313); pCH47 (N85-A85); pCH48 (N85-A85; N313- A313); pMW3 (4WC::His). Figure 76. Apyrase substrate preference of recombinant 4WC glycosylation mutants, determined from whole cell extracts. 4WC = wild type recombinant protein ; pCH45 (N313-A313); pCH47 (N85- A85); pCH48 (N85-A85; N313-A313); pMW3 (4WC::His).

Figure 77. SDS-PAGE/immunoblot analysis of fractions collected from mucin columns loaded with 4WC glycosylation mutants at various pHs. CE = crude extract; FT = flow through; LW = last wash; E = eluate. 4WC = wild type recombinant protein ; pCH45 (N313-A313); pCH47 (N85-A85); pCH48 (N85-A85; N313-A313).

Figure 78. Apyrase activity (square blocks) and protein concentration (diamonds) analysis of Size Exclusion Fraction assay of 6RG, 4WC apyrases and 4WC::6RG and 6RG::4WC chimeras.

BRIEF DESCRIPTION OF SEQUENCE LISTING

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention will now be more fully described with reference to the accompanying examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLES

CLONING APYRASES

The following is a brief description of the methodology used. Detailed information is available through the manuals and protocols provided by the manufacturers of the respective kits used. General molecular biology procedures, e.g. restriction digests etc., were performed according to protocols available in sources like Sambrook et al., (1989).

Putative LNP apyrase clones were chosen from Trifolium repens (white clover) and Lolium perenne (ryegrass) EST libraries by BLAST (Altschul et al., 1990) searching the EST databases using translated exon sequences from public domain apyrase sequences. *^■'.

The plasmids containing the ESTs (prepared in-house, using a standard alkaline lysis protocol) were transformed into electro-competent E. coli DH5α cells. Colonies were selected and alkaline lysis/PEG8000 plasmid extraction performed. Digests with EcoRI were used to check insert size. Sequencing initially was done with T7 and SP6 primers, subsequent oligonucleotide primers were designed to the initial sequence to resolve the entire length of insert.

Sequence alignment analysis

Multiple sequence alignments of 44 translated full length, 3 translated EST plant GDA1/CD39 apyrases and CD39 were generated with the Clustal implementation within MegAlign (DNASTAR, Madison, Wl), with manual optimization. Maximum parsimony analyses were performed with PAUP* 4.0b10 (Swofford, 2002) using heuristic searches with tree bisection reconnection (TBR) swapping. Gaps were treated as missing data. Bootstrap values were obtained with 100 random addition sequence replicates and TBR swapping (Figure 2).

N-terminal sequence data (where available) was analysed by TargetP V1.0 (Emanuelsson et al., 2000) and SignalP V2.0 (Bendtsen et al., 2004.). The predictions from these analyses were summarised and are presented in Figure 2.

Three clones containing full length Open Reading Frames (ORFs) were selected, namely: 4WC, 7WC and 6RG (Figure 1).

These full length clones were selected based on their clustering along with their predicted sub-cellular targeting and potential cleavage site of the signal sequence. The white clover clones (4WC and 7WC) align closely with the predicted secretory apyrases Db-LNP (Accession No. AF139807) and Lj-LNP (Accession No. AF156780) that are involved in the establishment of symbiotic relations possibly through cell-cell interaction. 7WC belongs to a clade containing sequences from only indeterminate nodule forming legume species while 4WC belongs to a clade that contains only legume sequences but from both determinate and indeterminate nodule forming species (Figure 2). Both Db-LNP and Lj- LNP are from determinate nodule forming legumes and have been shown to have roles in establishing legume/rhizobium and legume/mycorhizzal symbioses (Etzler and Murphy 2002; Etzler and Roberts 2001 ; 2005). Furthermore, Kalsi and Etzler (2000) have shown that Db-LNP is a peripherally membrane bound protein. The ryegrass clone 6RG was chose as it contains secretory elements and therefore has the potential to be involved in cell-cell interaction at the cell surface. In addition, demonstration of carbohydrate binding properties of apyrases that lie outside the previously identified LNP clade would expand the number of starting lectins for development. This has recently been confirmed by Fonseca et al., (2006) who demonstrated that Trypanosoma rangeli ecto-ATPase had elevated apyrase activity in the presence of specific carbohydrates.

METHODS OF PRODUCING POLYCLONAL ANTIBODIES TO RECOMBINANT APYRASES

Generation of polyclonal antiserum against truncated C-terminal 6RG apyrase

Not all predicted mature apyrase peptides are suitable for generating antibodies. In such cases we have shown that the C-terminal half of an apyrase can be sufficient to generate suitable antibodies. Two rabbits failed to generate antibodies against the mature recombinant 6RG peptide. For 6RG we used the 237 C-terminal amino acids of the 6RG peptide fused to a C-terminal 6xHis tag. The truncated recombinant protein was generated by cloning a 711 base pair section of the C-terminal of 6RG into the pET28 expression vector to generate the clone pCH25 (Figure 3).

A culture of BL21 E. coli cells containing the pCH25 construct was initiated and incubated at 37°C.

When the culture had grown to an OD₆oo of 0.6-0.7, expression of C-terminal 6RG was induced by the addition of isopropyl β-D-thiogalactopyranoside (IPTG ), and the culture was incubated for a further 3h at 37°C. The culture was harvested and inclusion bodies isolated. Samples taken during the induction period and a sample from the inclusion body preparation were analysed by SDS-PAGE/coomassie stained gel (Figure 4).

Proteins from the inclusion body prep were solubilised in buffered 6M urea and loaded onto a Nickel Affinity Column. Selected fractions were dialysed against phosphate buffered saline (PBS) and concentrated to 0.35μg/μL by lyophilisation. These fractions were further purified by whole gel elution (BIO-RAD) from an SDS-PAGE/coomassie stain gel (Figure 4).

Sufficient quantities (3-400μg) of the truncated C-terminal 6RG protein was purified using a 3 step process utilizing: inclusion body preparation; affinity purification (Ni²⁺ column for His tag) and gel elution. 300 μg of purified protein was injected into a rabbit over a period of 3 injections (100μg/injection + adjuvant) over 4 weeks to stimulate the generation of anti-apyrase polyclonal antibodies. Prior to immunisation a 'pre-bleed' blood sample was removed from the rabbit for comparison of specificity between pre- and post-immunisation antibody titres.

Generation of polyclonal antiserum against 4WC and 7WC apyrase

The sequences encoding the complete mature peptides of 4WC and 7WC were separately cloned in frame into into the pET28 expression vector providing C-terminal His tag fusions (Figures 5 & 6 respectively) and transformed into BL21 E. coli cells for recombinant protein expression. Induction, localisation and size determination studies were performed for each construct. In each case the protein was the predicted size (based on SDS-PAGE/coomassie) and was targeted predominantly to the inclusion bodies (Figures 7 and 8 respectively). Sufficient quantities (3-400μg) of each of the 4WC and 7WC protein products were purified using a 3 step process utilizing: inclusion body preparation; affinity purification (Ni²⁺ column for His tag) and SDS-PAGE whole gel elution (Figures 7 and 8 respectively). 300 μg of each purified protein was injected into separate rabbits over a period of 3 injections (100μg/injection + adjuvant) over 4 weeks to stimulate the generation of anti-apyrase polyclonal antibodies. Prior to immunisation a 'pre-bleed' blood sample was removed from the rabbit for comparison of specificity between pre- and post-immunisation antibody titres.

Analysis of antiserum against truncated C-terminal 6RG Rabbit polyclonal antibodies generated against E. coli expressed, C-terminal 6RG was analysed by SDS-PAGE/immunoblot-chemiluminesence detection of Horse Radish Peroxidase activity to determine titre and specificity. The maximum titre that was still able to detect 100ng (-0.0001 OD₂₈₀) of purified recombinant mature 6RG was between 1 :12,800 and 1 :25,000 (Figure 9). The maximum sensitivity by SDS-P AGE/immunoblot was 12.5ng using a 1 :1000 dilution (Figure 10). The pre-immune antibody serum has a negligible cross reactivity to recombinant 6RG.

Analysis ofantisera against 4WC and 7WC

Rabbit polyclonal antibodies generated against E. coli expressed, mature 4WC was analysed by SDS- PAGE/immunoblot to determine titre and specificity. The maximum titre that was still able to detect 100ng (-0.0001 OD₂₈o) of purified recombinant mature 4WC was between 1 :3,200 and 1 :6,400 (Figure 11 ). The maximum sensitivity by SDS-PAGE/immunoblot was 12.5ng using a 1 :500 dilution (Figure 12). Antiserum generated against 4WC is also able to detect recombinant 7WC but not 6RG (Figure 13).

Rabbit polyclonal antibodies generated against E. coli expressed, mature 7WC was analysed by immunoblot to determine titre and specificity. The maximum titre that was still able to detect 100ng (-0.0001 OD₂₈₀) of purified recombinant mature 7WC was between 1 :6,400 and 1 :12,800 (Figure 14). The maximum sensitivity by immunoblot was 25ng using a 1:500 dilution (Figure 15). Antiserum generated against 7WC is not able to detect recombinant mature 4WC (Figure 16).

This demonstrates that high affinity, high specificity anti-apyrase rabbit antibodies can be generated to as little as 300μg of purified, recombinant, mature apyrase or the C-terminal half of the apyrase.

METHODS OF ENHANCING EUKARYOTIC PRODUCTION OF HOMOGENEOUS RECOMBINANT APYRASE

Bacυloviral Expression

We have been successful in generating highly active recombinant soluble apyrase (7WC, 6RG, 4WC, 7WC::6RG, 6RG::7WC, 4WC::DbLNP, DbLNP::4WC, CD39) from E. coli (RECOMBINANT APYRASES AND METHODS FOR MAKING SAME patent, Chen, Scott, Cumming, Arcus, Roberts) we have also expressed some recombinant soluble apyrases (7WC, 6RG, 4WC, 6RG::4WC, 4WC::6RG) in a eukaryotic expression system in order to generate proteins with apyrase activity. The inability of prokaryotes to correctly perform eukaryotic post translational modifications is frequently blamed for the inactivity of some bacterially expressed recombinant proteins. C-terminal tagged and untagged full length 6RG, 7WC and 4WC apyrase clones were placed into the Baculo Direct Bacmid (Invitrogen) for expression and purification from insect cells (Table 1).

Table 1. Tagged and untagged full length constructs of 6RG, 4WC and 7WC for insect expression.

Cloning of 7WC±taq, 4WC±tag and βRG±tag Using specific primers (Table 2), PCR was used to generate fragments containing the full length open reading frame of 7WC₁ 4WC and 6RG; these incorporated the Gateway Adaptor sites for cloning into pENTR-D. To generate 7WC, 4WC and 6RG with a Thrombin-His fusion tag, specific PCR primers (Table 2) were designed to incorporate a Thrombin-His fusion tag at the 3¹ end of the 7WC, 4WC and 6RG ORFs. Two rounds of PCR were performed (Figure 17): the first round of PCR using a Gateway Adaptor forward primer and a Thrombin Adaptor reverse primer, and the second round of PCR using the same Gateway Adaptor forward primer and a Thrombin-His Adaptor reverse primer. The Thrombin-His Adaptor primer anneals at the thrombin cleavage site introduced at the 3' end of the first PCR product, allowing amplification from this primer to generate a final PCR product incorporating both the thrombin cleavage site and the His tag. These PCR products were then cloned into pENTR-D (Invitrogen).

The proofreading polymerase, Pfu (Promega), was used to amplify 7WC and 4WC using the primers outlined in Table 2. Ethidium bromide stained agarose gel analysis showed products of the correct size (Figure 18).

The ~1.4kb products (lane 2 for each) were purified using the QIAGEN QIAquick PCR purification kit. The two purified PCR products for 7WC-T and 4WC-T were then used as template in a second round of PCR to add a His tag, using the Thrombin-His adaptor primer, previously described (Table 2). Ethidium bromide stained agarose gel analysis showed products of the correct size (Figure 19).

A Taq (Invitrogen) PCR mix was used to amplify 6RG using the primers outlined in Table 2. Ethidium bromide stained agarose gel analysis showed product of the correct size (Figure 20). The ~1.4kb products (including 6RG with a 3" thrombin cleavage site, 6RG::thrombin) were purified using the QIAGEN QIAquick PCR purification kit. The purified 6RG:: Thrombin PCR product was then used as template in a second round of PCR to add a 3' His tag, using the Thrombin-His adaptor primer, previously described (Table 2). Ethidium bromide stained agarose gel analysis showed products of the correct size (Figure 21 ). Table 2. Primer sequences designed to amplify 7WC, 4WC and 6RG with or without 3' Thrombin/Thrombin-His tag, as specified. The thrombin cleavage site is indicated by italics and the His tag is the underlined region.

Construct PRIMER Sequence Xmer MW Tm(^°C) %GC pCH32/ENTRD-6RG 6RG Gateway Adaptor Fwd CACCATGGCTCACGCCATGATC 22 7248.6

73.9 59 _j. pCH32 6RG Gateway Adaptor Rev AATCACGACTCCTCGCTCAG 20 6326.0 64.5 55

PENTRD-6RG 6RG Thrombin Adaptor Rev ,ACr^CCGCGrGGGACGAGCGACTCCTGGCTCAGCTT 36 10959.2 87.2 64 pENTRD-βRG 3¹ Thrombin-His fusion TCAATGATGATGATGATGATGACCCGCGGAACTACCGCGrGGCyACCyAG 48 14826.3 91.754

PCH33/MW3 4WC Gateway Adaptor Fwd CACCATGGGTTTGCACTGGCC 21 6398.0 60.3 62 ^β pCH33 4WC Gateway Adaptor Rev CATGTCTGCACACATGTTAGCA 22 6694.3 64.7 45 pMW3 4WC Thrombin Adaptor RevΛCLACCGCGrGGCΛCGAGMTAAMTACATGAMCGATCAMTTTAGG 48 14794.5 80.940 pMW3 3' Thrombin-His fusion TCAATGATGATGATGATGATGACCCGCGGAACTACCGCGrGGGACGAG 48 14826.3 91.754 pMW1/2 7WC Gateway Adaptor Fwd CACCATGGAGTTCCTAATTAAACTCATCACA 31 9407.0 60.4 39 pMW1 7WC Gateway Adaptor Rev AGGCAAAGTTGGCTTCTAGTTATATCT 27 8304.2 60.3 37 pMW2 7WC Thrombin Adaptor >ACr>ACCGCGrGGGACGAGAACAAAATACATCAATCGTTCAAATTT 45 13749.8 61.040 pMW2 3' Thrombin-His fusion TCAATGATGATGATGATGATGACCCGCGGAACTACCGCGrGGGACGAG 48 14826.3 91.754

Cloning of 7WC±tag, 4WC±tag and βRG±tag into pENTR-D

The 7WC-throm bin/his tag (TH), 4WC-TH and 6RG-TH PCR products generated in the second round of PCR were purified using the QIAGEN QIAquick PCR purification kit. The six purified PCR products (7WC, 4WC and 6RG±tag) were then cloned into pENTR-D to generate pMW1 , pMW2 and pMW3 (Figures 22 & 23) and pCH32, pCH33, pENTRD-6RG (Figure 24), and pCH33 (Figure 25) using the protocols outlined by Invitrogen.

Four colonies from each transformation were minipreped using QIAprep® Spin Miniprep kit (Qiagen) and the purified plasmid analysed by using restriction enzymes known to generate specific sized fragments when used to digest the correct pMW1 , pMW2 or pMW3 plasmids (Figure 26 and Figure 27).

The plasmid lines were sequenced from the 5' and 3' ends using the primers indicated in Table 3. The sequences obtained showed that the 7WC, 4WC and 6RG had been cloned into the pENTR-D vector (Invitrogen), and that the 3' Thrombin-His tag was present and correct for pMW2 and pMW3 and pENTRD-6RG.

The pMW1 , pMW2, pMW3, pENTRD-6RG, pCH32 and 33 plasmid lines were linearised prior to the transfer by digesting with restriction enzyme Eco RV (Figure 28).

A phenol/chloroform extraction was carried out to purify the linearised plasmids. LR reactions were then carried out to transfer 7WC+taq, 4WC±tag and 6RG±tag from pENTR-D plasmids (pMW1, pMW2, pMW3, pCH32-33 and pENTRD-6RG) into the BaculoDirect C-terminal linear DNA. The resulting bacmids were named as per Table 1.

Insect cell/baculovirus expression of recombinant apyrases

Each of the bacmids were transfected into the insect cells Cellfectin® Reagent assisted lipid-mediated transfection and cultured in spinner flasks. Virus titre was amplified to the desired density through repeated passages of infection and culture. Expression of recombinant apyrase protein (7WC, 4WC or 6RG) was determined by SDS-PAGE/immunoblot for each of the bacmids.

Each of the bacmids were taken through to third passage in the insect cells and were of sufficiently high titre for subsequent protein expression studies.

Construct PRIMER Sequence Xmer MW TmCQ %GC

PCH32/33 pMWl, pMW2, pMW3 M13 Fwd GTAAAACGACGGCCAG 16 4924.2

57.6 56 pENTRD-6RG pCH32/33 pMWl-3 M13 Rev CAGGAAACAGCTATGAC 17 5212.4 50.6 47.1 pENTRD-6RG pENTRD-6RG 6rglSS gene specific TGTCCACACCAAGAGCAA 18 5461.5 61.5 50 pENTRD-6RG Apyrase 6rg new fwd 3 TGTGAGACGAGGAACTGCAC 20 6190.9 64.2

55 pENTRD-6RG 6RGlSs rev seq primer TTGCTCTTGGTGTGGACA 18 5536.5 61.6 50 pENTRD-6RG 6rgls revn CTCTCTGCATCTCTCGTACG20 5994.8 60.3 55 pMW3 pENTR 4WC N-His Fwd CACCATGGAAACAGTTACCTCGTACGCTG

29 8846.6 74.2 52 pMW3 4WC-RT Rev GGTATTCAATTTCATTTGCC20 6376.9 58.3 35 pMWl, pMW2 7WC3FS Gene Specifc Fwd TAGACTTGGGGCAACTGC 18 5843.7 61.4 55 ρMWl, pMW2 .;- 7WC-RT Rev TTTCTCCCCTGCTGTAATT 19 6303.9 58.7 42

Table 3 Primer sequences designed to sequence the pMW1 , pMW2, pMW3, pENTRD 6RG, pCH32 and pCH33 plasmid lines.

The constructs generated and taken to third passage in the baculovirus/ insect cells system are summarised in Table 4.

Apyrase done ffng Apyrase _{Jag p3} ^Pr .„„

pMW1 pMW-bacmid i 7WC none V V 3.8 X 10⁷

..,.,_ , »,., . . . „ -,,,_Λ C-term thrombin > pMW2 pMW-bacmιd 2 7WC _{deavage site &} V V 2.0 X 10⁷ pCH33 pCH-bacmid 1 4WC none V V 2.0 X 10⁸

„.„. . .... , . . _ ...,_ C-term thrombin , pMW3 pMW-bacmιd 3 4WC _c|_eavage site & ^V 1.5 X 10⁸ pCH32 pCH-bacmid 3 6RG none V V 3.0 X 10⁷

PENTRD6RG pCH-bacmid 4 6RG gST ^ V 3.3 X 10⁷

Table 4. Expression of the three recombinant plant apyrase constructs and P3 viral stock titre. P3 = viral stock isolated at third passage.

Protein was expressed by infecting fresh cultures of Sf9 insect cells with multiplicity of infection (MOI, defined as the number of virus particles per cell) of approximately 2. The transfected cultures were cultured in Complete TNM-FH (Grace's Insect Medium supplemented with yeastolate and lactalbumin hydrolysate and with the addition of 10% foetal bovine serum) in spinner flasks with paddles (stirring at ~60rpm). Where necessary, samples were removed at regular periods for analysis of expression levels.

The effect of time (post infection) on the production of maximum recombinant protein production was analysed by SDS-PAGE/immunoblot for each construct. In each case the maximum accumulation of recombinant protein was seen between 4-5 days post infection.

SDS-PAGE/immunoblot analysis of media extracts as well as whole cell extracts showed that the recombinant 6RG apyrase accumulated almost exclusively as one form in the cell extract (Figure 29). In comparsion, recombinant 4WC and 7WC accumulated as two forms in the cell and one form in the media (Figure 30, 31 and 32). For each clone the two forms in the cells were seen as two immunoreactive bands with different migration patterns in SDS-PAGE, where the faster migrating immuno reactive band was the same size as the media located protein secreted into the media. We found both forms of 4WC had the same amino acid sequence by trypsin digestion-electrospray-MS analysis. We have shown that the difference in migration patterns is due to differential glycosylation and we have exploited this to provide a method of isolating recombinant apyrase with uniform glycosylation (described later).

METHODS OF PURIFYING RECOMBINANT SOLUBLE APYRASES

The purification protocols needed to be tailored for each recombinant apyrase depending on: the clone; protein localisation and the presence or absence of a His tag. We demonstrate below that a C- terminal His tag can be used to aid purification of the soluble recombinant apyrases and does not need to be cleaved in order to show apyrase activity. Table 5 summarises the suitability of different purification methods for each recombinant apyrase; the results are discussed below.

A number of methods were used in this study to purify 4WC, 7WC and 6RG. Protein of high quality was isolated from 6RG::His using Nickel-Affinity column purification and from 4WC pellets and medium using a mucin column to purify the protein. A single step using NTA columns purified 6xHis tagged 6RG LNP to a >10-fold increase in purity and concentrated it to to >75% of the total protein. With 4WC, each of the purification steps tested increased the relevant purity of 4WC. Details of these methods are outlined below.

Constructs Method(s) Protein Quality

6RG::His Nickel-Affinity column •/S

4WC medium Mucin column, ion exchange ✓V

4WC pellets Mucin column •/

4WH^1-HiR medium Ninkfil-Affinitv r.nh imn V

4WC::His pellet Mucin column, Nickel column *

7WC medium Mucin column *

7WC pellets Mucin column *

7WC::His-E coli Nickel-Affinity column ✓✓

Table 5. Purification of proteins from different sources

SS = highly enriched, ^•/ = some enrichment, * = little or no enrichment

Ammonium sulphate precipitation of proteins

The culture media or cell pellet lysate was transferred into a clean beaker or spinner flask and placed on a magnetic stirrer in a cold room and allowed to equilibrate to 4°C over ~1 h. Ammonium sulphate was then gradually added over ~10min to give the desired level of saturation. The mixture was stirred for 1h, transferred into centrifuge tubes and spun at either 4080xg 15min 4°C for FPLC analysis, or

15,000xg 30min 4°C for mucin column binding analysis. The supernatant was transferred back to the beaker or flask, and returned to the magnetic stirrer in the cold room. The process of adding ammonium sulphate, mixing and spinning in a centrifuge was repeated to acquire the desired saturated ammonium sulphate fractions.

For some applications the resulting protein precipitate was resuspended in the target buffer then dialysed at 4°C against 100-1000 volumes of the target buffer, with a minimum of three changes of buffer. Extraction of cell localised recombinant protein for Ni-NTA purification

Insect cells were harvested and lysed as above. The supernatant was collected and loaded onto a Ni²⁺-agarose column, which was then washed with Binding Buffer (2OmM Tris.HCI pH7.9, 50OmM NaCI₁ 5mM Imidozale, 5mM β-mercaptoethanol, 5% glycerol) and followed by Wash Buffer (2OmM Tris.HCI pH7.9, 50OmM NaCI, 2OmM Imidozale, 5mM β-mercaptoethanol, 5% glycerol) to remove non-bound proteins. His tagged protein was eluted from the column using Elution Buffer (2OmM Tris.HCI pH7.9, 50OmM NaCI, 25OmM Imidiazole, 5mM β-mercaptoethanol, 5% glycerol) in 1mL aliquots. The purified recombinant 6RG was visualised by SDS-PAGE/coomassie stain and SDS- PAGE-immunoblot analysis.

In comparison to 6RG+tag, 4WC+tag and 7WC+tag from the insect cell pellet showed comparatively poor enrichment using Ni-NTA purification (Figures 29, and 30). Where Ni-NTA enrichment of 4WC+tag from the medium was moderately successful (Figure 31 ).

Purification of6RG::His by nickel column

Four-day post-infected insect cells were harvested by centrifugation at 2,000*g for 5min, and the pelleted cells washed twice with 1 xPBS, then resuspended and lysed in Extract Buffer (10OmM NaPO₄ Buffer pH7.5, 50OmM NaCI, 1 mM PMSF, 1 % Nonodet P-40). The soluble protein mixture was then clarified by centrifugation at 39,000*g for 30min. Imidazole was added in the supernatant to a final concentration of 5m M.

The precharged Ni²⁺-NTA resin (Invitrogen) was packed on the column and equilibrated with Binding Buffer (2OmM Tris-HCI pH7.9, 50OmM NaCI, 5mM Imidiazole, 5mM β-mercaptoethanol, 10% glycerol).

The supernatant was loaded onto the Nickel-Affinity column, washed with a minimum of 10 bed volumes of Binding Buffer (2OmM Tris-HCI, pH 7.9, 50OmM NaCI, 1OmM imidiazole) followed by a minimum of 10 bed volumes of Washing Buffer (2OmM Tris-HCI pH7.9, 50OmM NaCI,

2OmM Imidozale, 5m M β-mercaptoethanol, 10% glycerol), eluted with 1 * Elution Buffer (2OmM Tris-HCI pH7.9, 50OmM NaCI, 25OmM imidiazole, 5mM β-mercaptoethanol, 10% glycerol).

The eluate was collected as 500μl_ fractions and the protein concentration of each fraction was determined using the Bradford Assay (Bio-Rad). The fractions containing the highest protein concentration were pooled and dialysed against Apyrase Assay Buffer (either 5OmM MOPS pH6.8 or 5OmM Tris-HCI pH8.0, and 10% glycerol). After dialysis, the protein concentration was determined by Bradford Assay using BSA as a standard and stored at -80°C until required.

Selected samples were prepared for separation by SDS-PAGE, and protein content screened by coomassie staining and immunoblot analysis,

The recombinant 6RG::His purified by Nickel-Affinity column was enriched over 100-fold. SDS-PAGE- immunoblotting demonstrated that the major bands were 6RG::His. The two bands in both the Coomassie stained gel and the immuno blot are due to variation in glycosylation patterns (Figure 29).

Overall Expression and Purification Strategy for Recombinant 6RG

We have developed a protocol using recombinant full length 6RG::6xHis expressed using the baculoviral system. An outline of this protocol is shown as a flow chart in Table 6.

Infect insect cells with relevant construct, grow for 4 days i Harvest by centrifugation (2,000xg, 5m in) i

Wash pellet twice with IxPBS and resuspend in extraction buffer (10OmM NaPO₄ Buffer, pH7.5, 50OmM NaCI, 1 mM PMSF, 1 % Nonodet P-40)

Clarify the soluble protein by centrifugation (39,000xg, 30min). Add imidazole to the supernatant to a final concentration of 5mM

4,

Load in Binding Buffer onto a precharged a Ni²⁺-NTA column; Wash with wash buffer; Elute with elution buffer containing 25OmM Imadiazole; Dialyse with Tris or MOPS Buffer

Concentrate active protein using Amicon 10 columns (Millipore); Run through a size exclusion column and collect fractions; Analyse fractions for activity

I Re-concentrate active fraction by Amicon 10 column

Table 6. Flow chart for expression and purification of recombinant 6RG::His in insect cells. Recombinant full length 6RG::6xHis was run through a HiPrep Sephacryl S-100HR (Amersham) size exclusion column. The eluent showed that the active recombinant protein eluted from the column at approximately the same time as a 58kDa standard (Figure 33 and Table 7). These results indicate that the highly purified recombinant active glycosylated 6RG exists in a monomeric form (the recombinant mature non glycosylated protein is predicted to have a monomeric size of 48.9kDa).

Activity

Fraction Protein [Pi] (μmol Pi/ min/

(ng/uL) mg protein)

31 2 -0.1 0*

32 0 -0.3 0

33 15 0.2 1

V-Ti 34 0 -0.2 0

35 59 1.0 2

36 114 7.2 6

37 79 13.5 17

38 68 12.7 18

39 17 15.5 91

40 18 12.2 66

41 38 76.7 197

42 90 104.2 113

43 128 105.3 81

44 119 104.6 86

45 123 108.8 87

46 73 108.3 145

47 28 75.3 268

48 8 18.1 225

49 1 r ¹n²⁰, ,

exclusion column. 10μL of each fraction was taken to assay apyrase activity.

Mucin Column Purification of 4WC

The properties of LNPs include both apyrase and carbohydrate binding activity. Since 4WC::His was comparatively poorly enriched through nickle colums we were able to enrich the recombinant mature 4WC (no tag) protein by utilising its carbohydrate binding properties to capture the protein on a mucin column. The mucin column contains a mixture of carbohydrates that are bound to CNBr-activated sepharose. Carbohydrate binding is first determined by binding to an immobilised complex carbohydrate matrix derived from porcine A + H blood group substance. We used a commercially available complex carbohydrate mixture (i.e. gastric mucin). A carbohydrate column was generated by coupling gastric mucin III from porcine stomach (Sigma-Aldrich, Cat # M1778) to CNBr-activated sepharose (Amersham Biosciences). Typically the coupling efficiency was estimated to be 1.1 mg protein / g freeze dried CNBr-activated sepharose. This is calculated on the assumption that mucin is 10-20% protein and 80-90% O-linked carbohydrate which equates to 5-1 Omg of O-linked carbohydrate / g freeze dried CNBr-activated sepharose. LNPs bind specifically to target carbohydrates while the majority of the other proteins from the cell extracts or media are washed from the column.

Expression of 4WC in insect cells typically resulted in recombinant protein being localised in both the medium as well as in the cell. Hence, the optimum pH for mucin binding and eluting 4WC from both^ cell lysate and the media was determined independently. Insect cell/baculovirus lysate was equilibrated against Buffer A adjusted to a range of pH's (10OmM PO₄ Buffer adjusted to a pH of either 3.0, 4.5, 6.0, 7.5 or 9.0). Extracts were cleared by centrifugation and the supernatants were loaded directly onto 1mL columns containing pre-equilibrated mucin bound to sepharose. The columns were washed with the relevant Buffer A until no further protein was removed. Elution was performed by the inclusion of 0.5M NaCI in the relevant Buffer A (i.e. relevant pH).

Mucin purification of 4WC located in the media

To increase the relative concentration of 4WC in the initial cell media, an ammonium sulfate precipitation step was included prior to passing the samples through the mucin column.

Media from 96h post-transfection cultures was prepared, as outlined above. The samples were loaded and eluted from the mucin columns at a range of pH's. SDS-PAGE/silver stain analysis revealed that

, binding and elution at pH 9.0 of 4WC from the media results in maximum purity - in terms of the percentage of 4WC to other proteins (Figure 34). However, the amount of 4WC eluted from the column is lowest at pH 9.0, as shown by the lower intensity of the band on the SDS-PAGE/immunoblot (Figure 35). It appears that 4WC located in the media does not bind to the mucin at pH 3.0.

Mucin purification of 4WC located in the Cell

Cell lysate from 96h post-transfection cultures was prepared, as outlined above. The samples were loaded and eluted from the mucin columns at a range of pH's. The SDS-PAGE/silver stain analysis revealed the purity of 4WC in the eluates increases as pH decreases to pH 3.0 (Figure 36). However, the immunoblot shows that maximum recovery occurs at pH 7.5, as seen by the SDS- PAGE/immunoblot (Figure 37). At pH 4.5 the lack of a band in the crude extract (CE) sample indicates that 4WC from the cell lysate precipitates at this pH.

Overall Expression and Purification Strategy for Media Localised, Recombinant 4WC

We have developed a protocol using recombinant 4WC expressed using the baculoviral system. An

F\ outline of this protocol is shown as a flow chart in Table 8.

Infect insect cells with relevant construct, grow for 5 days i Remove cells by centrifugation (200Og, 5min)

1

Precipitate media using 60-80% ammonium sulphate cut at 4⁰C, centrifuge (39,000*g, 30min);

Resuspend precipitate in Extract Buffer (5OmM Tris HCI, pH8.0, 10OmM NaCI and 10% glycerol);

Dialyse against 1OmM NaPO₄ pH9.0 containing 10% glycerol; Clear debris by centrifugation

(39,000^χg, 30min) i

Load in binding buffer onto a mucin column pre-equilibrated with equilibration buffer, 1OmM NaPO₄, pH9.0, 10% glycerol

I

Wash column extensively with 1OmM NaPO₄, pH9.0, 10% glycerol, then elute with elution buffer (1OmM NaPO₄, pH9.0, 50OmM NaCI, 10% glycerol)

Dialyse active eluted fractions against 5OmM Tris-HCI pH8.0 containing 10% glycerol

4,

Load onto Ion Exchange column equilibrated with Load Buffer (25mM Tris-HCI pH7.5, 10% glycerol,

20μM PA and 4mM DTT); Wash column with Load Buffer; Elute column with Elution Buffer (Load

Buffer + 1M NaCI), collect 1mL aliquots for each fraction

4^, Pool and concentrate active fractions by Amicon 10 column i Fractionate by size exclusion column, collect fractions and analyse apyrase activity

Re-concentrate active fraction by Amicon 10 column

Table 8. Flow chart for expression and purification of media localised recombinant 4WC from insect cells. The combination of precipitating protein from the media followed by mucin column then ion exchange column purification steps yielded a highly purified protein seen as a single band on a silver stained SDS-PAGE gel (Figure 38).

The recombinant 4WC protein from medium was enriched approximatelyl 0-fold as it was resuspended in extract buffer of one tenth the original volume after ammonium sulphate precipitation. The SDS-

PAGE/silver stained gel suggested that 4WC was the major protein in the eluates collected from the

Mucin column; this was confirmed by lmmunoblot analyis. It is estimated that the recombinant 4WC was further enriched approximately 100-fold at this step. Therefore, in combination with the ammonium sulphate precipitation, the recombinant 4WC protein from medium was enriched approximately 1 ,000-fold.

Under the ion exchange conditions used, 4WC passed through the column during loading, while the 'contaminating' proteins were captured on the column. Following Ion Exchange chromatography 4WC is represented as a single band even with the higher sensitivity of silver staining. The total enrichment of 4WC from the medium for all the purification steps is approximately 5,000-fold.

Purification of recombinant 4WC localised in the cell

The infected 4WC cell pellets were lysed using Lysis Buffer (10OmM NaPO4 Buffer pH7.5, 10OmM NaCI, 1% NP-40 and 5mM β-mercaptoethanol) then dialysed against Equilibration Buffer (1OmM NaPO4 Buffer pH3.0). The solution was cleared by centrifugation at 39,000*g for 30min. The supernatant loaded onto a mucin column pre-equilibrated with Equilibration Buffer and washed extensively with Equilibration Buffer. 500μl_ fractions were eluted from the column with Elution Buffer (1OmM NaPO4 Buffer pH3.0, 50OmM NaCI) and analysed by SDS-PAGE/silver stain and immunoblot gels (Figure 39).

The recombinant 4WC from cell pellets was enriched by at least 100-fold, as no 4WC could be detected by SDS-PAGE/immunoblot in the crude extract. From the silver stained gel, 4WC appeared to account for approximately 50% of the total protein content (Figure 39). Purification of 4WC::His tag proteins from cell pellets and medium

The supernatant containing the recombinant protein 4WC::His was crudely fractionated by increasing the concentration of ammonium sulphate to precipitate proteins. The recombinant 4WC was collected in the 60%-80% ammonium sulfate fraction. The precipitate was resuspended in Extract Buffer (5OmM Tris-HCI, pHδ.O, 10OmM NaCI and 10% glycerol) and dialysed against the same buffer. The debris was cleared by centrifugation at 39,000*g for 30min. Subsequent Nickel-Affinity column purification of 4WC::His from both the ammonium sulphate fractionated culture medium and the cell pellet, steps were the same as previously described for 6RG::His. The results of analysis by SDS-PAGE/silver stain and immunoblot are shown in Figures 30 and 31. These results show that the recombinant protein 4WC::His from medium and cell pellets could be purified by Nickel-Affinity column, although the purity of 4WC::His obtained from the two sources was quite different. It is estimated that greater than 50% of the protein purified from the medium is 4WC::His, and there is only a limited number of contaminating proteins (as seen on the silver stained gel). In contrast, only 30% of the total protein purified from the cell pellets is 4WC::His, and there are a large number of contaminants.

METHODS OF MANIPULATING SOLUBLE APYRASE MUCIN BINDING, SUBSTRATE PREFERENCE, pH OPTIMUM AND INHIBITOR INTERACTION

Apyrase Activity Analysis

Xi

In general, reactions were in a total volume of 100μL, containing 5OmM Buffer, 5mM Substrate, 3mM CaCI₂, and target protein; the reaction was incubated at 25^°C for 30min then the inorganic phosphate was measured. Recombinant apyrase was assayed for apyrase activity using the following protocol: affinity purified recombinant apyrase was dialysed against 6OmM MOPS (pH6.8); 20ng of the purified recombinant apyrase was then mixed with 100μL Assay Buffer (6OmM MOPS Buffer pH6.8, 1mM MgCI₂, 3mM ATP (or other nucleotide tri- or di-phosphate)) and incubated at 25°C for 30 min. 30μL of the mix was transferred to a microtiter plate, and 125μL Reaction Mixture (4 parts Ammonium Molybdate Reagent [15mM Zinc acetate, 1OmM Molybdate (added to zinc acetate), pH adjusted to 5.0 with cone HCI] to 1 part Reducing Agent [prepared fresh, 10% ascorbic acid, pH adjusted to 5.0 with NaOH]) was added, mixed, and incubated at 3O⁰C for 10 minutes. Absorbance at λ = 630nm was determined using a plate reader. Apyrase activity such as 700U for 7WC::His (one unit is defined as 1 μmol Pi released by apyrase in one minute per mg protein), i.e., 700 μmol Pi/min/mg 7WC was calculated from the quantity of free phosphate released by comparing absorbance with inorganic phosphate standards (10, 20, 40, 60, δOnmol PO₄ ^", pH6.8).

Effect ofpH on apyrase activity of 4WC and 4WC::His from the medium

A series of assays were performed using 5mM ADP as the substrate, in different buffers with pH values increasing in steps of 0.5. The buffers used here are 5OmM Sodium Acetate Buffer (pH3.0, 3.5, 4.0, 4.5 and 5.0), 5OmM MES (pH5.0, 5.5, 6.0, 6.5 and 7.0), 5OmM MOPS (pH6.5, 7.0, 7.5 and 8.0) and 5OmM Tris-HCI (pH8.0, 8.5, 9.0 and 9.5). The results are shown in Figures 40 and 41.

The results indicate that both 4WC and 4WC:: His possess apyrase activity in a wide range of pH values (3.0-9.5), including very acetic conditions, such as pH3.0. 4WC and 4WC::His apyrase activity was highest at pH9.5 in Tris-HCI Buffer.

Substrate preference of 4WC and4WC::His

A range of substrate preference experiments was carried out using 4WC in Tris-HCI Buffer pH9.5 containing 3mM CaCI₂, with 4WC and a range of NTPs and NDPs at 5mM. A parallel control was run for each sample without the inclusion of enzyme. Each of the enzyme activities were calculated by subtracting the value observed for the corresponding control sample (Figure 42).

The results indicate that 4WC utilises both NDPs and NTPs as a substrate, with a preference for NDPs. The substrate preference in decending order is:

TDP> GDP> CDP> UDP> ADP≥ CTP> UTP> TTP> GTP> ATP.

Using similar conditions, the substrate preference for ATP or ADP by 4WC::His was compared (Figure 43). The results were very similar to native 4WC in that ADP was the preferred substrate with approximately the same ATP::ADP ratio/preference for both 4WC and 4WC::His. The addition of the C-terminal His tag did not affect the preference for ADP compared with ATP. Substrate binding site analysis for 4WC

In order to determine the 4WC binding sites for NTPs and NDPs a series of experiments were performed in which the amount of either ATP or ADP was serially increased either as the only substrate, or concomitantly with a constant level of the other nucleotide (i.e. ATP and ADP together). Four groups of substrate combinations were studied (described in Table 9)

Group Substrate (mM)

1 ATP 0.25 0.50 0.75 1.00 2.50 5.00 7.50

2 ADP 0.25 0.50 0.75 1.00 2.50 5.00 7.50

ATP 5.00

³ ADP-

0.25 0.50 0.75 1.00 2.50 5.00 7.50

V

ATP-

0.25 0.50 0.75 1.00 2.50 5.00 7.50

4 V

ADP 5.00

Table 9. Concentrations of ADP, ATP and ADP/ATP used in competition experiment.

The results of the substrate competition study indicated that when the ADP concentration was fixed (5mM) and the ATP was varied, the activity declined as the ATP concentration increased (Figure 44). Whereas when the ATP concentration was fixed (5m M) and the amount of ADP in the assay was varied, the activity increased as the ADP concentration increased. Hence, 4WC preferentially utilised ADP as a substrate and both ADP and ATP likely bind the same active site.

Binding at the same site can be demonstrated by showing that the total velocity is independent of the ratio of the two substrates (Chevillard e£a/.,1993). This type of analysis can become somewhat problematic for apyrases since some apyrases are able to utilise both NTPs and NDPs as substrates. Hence the dephosphorylation of a NTP to its respective NDP presumably leaves the NDP already bound or in close proximity to the active site; thus the original NTP may effectively occupy the same site for two separate catalytic reactions. Furthermore, the apyrase may have different catalytic rates for NTPs and NDPs, hence if their binding affinities are similar the rate of catabolism will also influence the amount of time the substrate occupies the site for. Alternatively, the catalytic rates may be similar but the binding affinities may be different; hence the addition of the low affinity substrate will have little influence over the overall reaction. Whereas addition of high affinity substrate will change the reaction considerably. In the case of 4WC it appears that the binding and cleavage of ATP to ADP is the rate limiting step, hence we have assumed that once bound ATP is cleaved twice to release AMP and 2 molecules of inorganic ortho phosphate. We used Chevillard's analysis to evaluate 4WC and began by choosing a common velocity of 4.5 which occurs with either 2.5mM ATP or 1.OmM ADP (determined from Figure 44). At these concentrations the substrates are considered to have a 1 :1 ratio since they result in a common velocity. The influence of substrate ratio on total velocity is then determined by varying the substrate concentrations over a range of ratios (P) 0:1 to 1 :0 (Table 10).

P=ADP/ATP 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

ATP(mM) 2.5 2.25 2.0 1.75 1.5 1.25 1.0 .075 .05 0.25 0

ADP(mM) 0 0.1 0.2 0.3 0.4 0.5 ^{" '} 0.6 0.7 0.8 0.9 1.0

Table 10. Ratio of the substrates ADP and ATP for competition with 4WC.

Under these conditions the maximum concentration of ATP (2.5mM) in the absence of ADP would result in a reaction velocity of 4.5; similarly for the maximum concentration of ADP (1mM) in the absence of ATP. When all conditions were tested we found that the activity was virtually independent of the ratio of ADP/ATP (Figure 45). This supports the claim that ADP and ATP bind to the same active site of 4WC. Effect ofpH on apyrase activity of 6RG:: His

This experiment was set up in the same way as described for the analysis of 4WC activity.

The activity of 6RG::His peaked sharply at pH6.5 when assayed with ATP as the substrate (Figure 46). No notable difference in activity was observed between different buffers used at the same pH. Subsequent studies of 6RG::His were carried out in 5OmM MES pH6.5.

Substrate preference of 6RG::His

The assay was performed as described for 4WC, using 5OmM MES Buffer pH6.5. The result revealed that 6RG::His has a very high preference for NTP substrates over NDPs (Figure 47).

The order of preferred substrates for 6RG::His is: GTP>ATP>CTP>UTP>TTP»>TDP>ADP>UDP>GDP>AMP.

Despite the additional His tag at C-terminus of 6RG the recombinant protein is still an active apyrase.

Substrate competition with 6RG::His

The assay setup was the same as for 4WC, using the four groups of substrate combinations (as described in Table 9). Similar apyrase activity curves were displayed for both ATP alone, and the varying ATP with constant ADP. 6RG showed virtually no apyrase activity with ADP alone whereas the addition of a constant level of ATP plus increasing amounts of ADP resulted in a constantly high apyrase activity which decreased slightly with increasing amounts of ADP (Figure 48). Considering 6RGs substrate preference the simplest explanation of this would be that ATP binds to the active site whereas ADP binds very poorly.

pH profile of 7WC::His

The pH profile activity of purified, active, recombinant 7WC::His from E. coli was set up in the same way as described for the analysis of 4WC activity. Activity was observed for all of the buffers tested, with maximum activity occurring at pH 8.0 (Figure 49). Substrate preference of 7WC::His

The substrate preference assay of purified, active, recombinant 7WC::His from E. coll was set up in the same way as described for the analysis of 4WC. The results indicate that while 7WC::His utilises both NTPs and NDPs as substrates, the protein shows a preference for NTPs (Figure 50). The substrate sequence order is:

ATP>TTP>UTP>CTP^«GDP=UDP>GTP>ADP^«TDP>CDP>AMP.

Substrate competition with 7WC::His

The assay setup was the same as for 4WC, using the four groups of substrate combinations as described in Table 9 with one additional substrate concentration, 10mM. The results of the competition study indicated that when the ADP concentration was fixed (5mM) and the ATP was varied, the activity was relatively constant; similarly, when the ATP concentration was fixed and the ADP was varied. However, in the latter case the level of activity was always lower than the corresponding ADP/variable ATP samples (Figure 51). Hence, 7WC preferentially utilised ATP as a substrate and both ADP and ATP likely bind the same active site.

Binding at the same site can be demonstrated by showing that the total velocity is independent of the ratio of the two substrates (Chevillard et a/., 1993). This type of analysis can become somewhat problematic for apyrases since some of them are able to utilise both NTPs and NDPs as substrates. Hence the dephosphorylation of a NTP to its respective NDP presumably leaves the NDP already bound or in close proximity to the active site; thus the original NTP may effectively occupy the same site for two separate catalytic reactions. Furthermore, the apyrase may have different catalytic rates for NTPs and NDPs, hence if their binding affinities are similar the rate of catabolism will also influence the amount of time the substrate occupies the site for. Alternatively, the catalytic rates may be similar but the binding affinities may be different; hence the addition of the low affinity substate will have little influence over the overall reaction. Whereas addition of high affinity substrate will change the reaction considerably. Since the apparent Vmax for 7WC appears to be roughly the same using either ATP or ADP as substrate, in the absence of non cleavable substrates it is difficult to determine which of these scenarios is correct. Nonetheless, we used Chevillard's analysis to evaluate 7WC and began by choosing a common velocity of 42.5 which occurs with either 1 mM ATP or 5mM ADP (determined from Figure 49). At these concentrations the substrates are considered to have a 1 :1 ratio since they result in a common velocity. The influence of substrate ratio on total velocity is then determined by varying the substrate concentrations over a range of ratios (P) 0:1 to 1 :0 (Table 11 ).

Table 11. Ratio of the substrates ADP and ATP for competition with 7WC.

Under these conditions the maximum concentration of ATP (1 mM) in the absence of ADP would result in a reaction velocity of 42.5; similarly for the maximum concentration of ADP (5mM) in the absence of ATP. When all conditions were tested we found that the activity was consistently greater than expected at all ratios of ADP/ATP (Figure 52). These results can be explained by 7WC binding to NTP, rapidly cleaving it to NDP then cleaving this to NMP without releasing the intermediate. Hence, the true binding site competition is obscured' by the fact that bound NTP rapidly becomes the competing substrate NDP occupying the binding site which is not in true competition with the remaining free NTPs. The apparent lack of competition becomes even more exacerbated if the rate of NTP phosphohydrolysis to to NDP is considerably greater than the rate of NDP phosphohydrolysis to NMP. Alternatively NTP binds very poorly but when it does it is rapidly cleaved to ADP which now competes with other ATP.

The pH profiles and substrate preferences of recombinant 4WC, 6RG::His and 7WC::His are sufficiently different to distinguish them by apyrase assay alone.

Mucin binding analysis Carbohydrate columns were generated by coupling gastric mucin III from porcine stomach (Sigma-Aldrich) to CNBr-activated sepharose (Amersham Biosciences). Typically the coupling efficiency was estimated to be 1.1 mg protein / g freeze dried CNBr-activated sepharose. This is calculated on the assumption that mucin is 10-20% protein and 80-90% O-linked carbohydrate which equates to 5-1 Omg of O-linked carbohydrate / g freeze dried CNBr-activated sepharose.

Crude soluble protein (containing recombinant 4WC, 7WC or 6RG) was either directly extracted from insect cells by grinding washed frozen cells in a variety of 10OmM buffers with the following pH values 3.0, 4.5, 6.0, 7.5, 9.0, or aliquots of the media were dialysed against the same buffers. Cells extracts were cleared by centrifugation and the supernatants were loaded directly onto 500μL columns containing pre-equilibrated mucin bound to sepharose. The columns were washed with the relevant extraction buffer until no further protein was removed. Elution was performed by the addition of NaCI (to 0.5M) to the buffer. Column eluent was fraction collected and visualised by OD₂so and SGS-PAGE coomassie stain and immunoblot analysis.

E. coli expressed, purified, refolded 7WC::His was also analysed for carbohydrate binding by the same technique.

Constructs compared for carbohydrate binding are shown in Table 12.

Expression

Gene Construct System

4WC pCH-Bacmid1 insect

6RG::His pCH-Bacmid4 insect

7WC pMW-Bacmid1 insect

7WC::His pET28-7WC bacteria

Table 12. Source of constructs used to analyse carbohydrate binding. Preparation of the 4WC sample from the cell pellet (4WC-P)

Five-day post infected insect cells were harvested by centrifugation at 3,000*g for 5min, and the pelleted cells washed twice with 1 *PBS. The cell pellet was resuspended in Extract Buffer (10OmM NaPO₄ Buffer pH7.5, 50OmM NaCI, I mM PMSF, 1% Nonodet P-40) and cleared by centrifugation at 39,000*g for 30min. The supernatant was aliquoted into five equal portions and dialysed against 1OmM NaPO₄ Buffer buffered at a range of pHs - 3.0, 4.5, 6.0, 7.5 and 9.0.

Any precipitate that had formed during dialysis was removed by centrifugation (11 ,000*g 5min 4°C). The supernatant was loaded onto the mucin column that had been equilibrated to the same pH as the supernatant with the relevant Equilibration Buffer (1OmM NaPO₄ Buffer at pHs - 3.0, 4.5, 6.0, 7.5 and 9.0). The column was extensively washed with Equilibration Buffer (>20 bed volumes), then eluted with Elution Buffer (1OmM NaPO₄, 50OmM NaCI, at the same pH as the supernatant). Samples were collected as they eluted from the column, including the flow-through fraction during loading, the last 500μL of the wash step, and 500μL fractions during the elution.

Preparation of the 4WC sample from the culture medium (4WC-M)

The supernatant from a five-day post infected insect cell culture was harvested by centrifugation at 3,000*g for 5min. The volume was measured and ammonium sulphate was slowly added to the solution with stirring to give a final concentration of 60% saturation at 4°C. The mixture was centrifuged at 10,000*g for 20min, the supernatant transferred to a fresh container and its volume remeasured. Ammonium sulphate was added to a concentration of 80% saturation at 4°C. After stirring at 4°C the precipitate was recovered by centrifugation at 10,000*g for 20min. The precipitate was resuspended in Extract Buffer (10OmM NaPO₄, pH7.5, 50OmM NaCI) to V₁₀ of original volume, divided into five equal parts and dialysed against 1OmM NaPO₄ Buffer buffered at a range of pHs - 3.0, 4.5, 6.0, 7.5 and 9.0. Mucin column purification was performed as above. SDS-PAGE/immunoblot analysis of4WC-P and4WC-M

The approximate protein concentration for each sample was determined by measuring the absorbance at 280nm. Samples from the mucin columns, including; crude extract (CE), flow-through (FT), last wash fraction (LW) and elution fraction containing the greatest protein concentration, were visualised by SDS-PAGE/immunoblot. Equal volumes of the various samples were loaded onto the SDS-PAGE gel. The protein was transferred to a PVDF membrane and immunoblot analysis was performed using the anti-4WC antibody (Figures 35 and 37).

4WC-P isolated from the |ysed cell pellets bound to the mucin column at pH3.0, 6.0, 7.5 and 9.0. 4WC-P preciptiated at pH4.5 which suggests that the isoelectric point may occur at this pH. At pH7.5, the presence of 4WC protein in the flow-through fraction may be the result of column overloading.

4WC-M from medium binds to the mucin at pH4.5, 6.0, 7.5 and 9.0. • At pH3.0, no 4WC-M was detected in the elution fraction indicating that at this pH it does not bind to the mucin column.

7IVC from insect cell pellets and medium

The isolation and mucin column purification of 7WC from the lysed cell pellet and culture medium followed the same protocols as described for 4WC, respectively.

Some enrichment of 7WC-M can be seen at pH 7.5 and 9.0. Optimum binding of 7WC-P to the Mucin column occurred at pH 6.0, with weaker binding seen at pH 4.5, 7.5 and 9.0. In a similar pattern seen with 4WC, two distinct bands of different molecular weights were observed in the 7WC-P samples at pH 7.5 and 9.0 (Figure 53).

Carbohydrate binding of 7WC::His from E. coli

E. coli expressed 7WC::His was purified using a Ni²⁺-NTA affinity column and was refolded as per the applicant's other patent applications, relating to Recombinant Apyrases And Methods For Making Same, based on New Zealand Patent Application No. 552725 and New Zealand Patent Application No. 552413 (Chen, Scott, Cumming, Arcus, Roberts). The refolded E. coli expressed active 7WC::His was also analysed for carbohydrate-binding in the same manner as the baculoviral expressed 7WC described above. The results of the Mucin column binding are shown in Figure 54.

The E. coli expressed 7WC::His Mucin binding results are similar to those seen when using the baculoviral cell pellet extract rather than the baculoviral medium extract. The E. coli expressed 7WC::His protein is predominantly able to bind between pH6-7.5. The lack of immunoreactive protein at pH4.5 is thought to be due to precipitation of the protein at this pH (similarly for 4WC from the cell pellet at the same pH).

6RG:: His from insect cell pellets

Since insect cell expressed 6RG::His is normally only located in the cell pellet and not in the medium the mucin binding analysis only dealt with the cell pellet/lysed cell fraction of the insect cell/baculovirus cultures. The isolation and mucin column purification of 6RG::His from the cell pellet followed the same protocols as described for 4WC-P pellet located protein.

In brief, the infected 6RG::His cells were harvested, resuspended in Extract Buffer, dialysed against 1OmM NaPO₄ Buffer buffered at a range of pHs - 3.0, 4.5, 6.0, 7.5 and 9.0, and clarified by centrifugation. The supernatant was then loaded onto a Mucin column prepared for analysing the effect of pH on carbohydrate-binding.. Selected samples were analysed by SDS-PAGE/immunoblot using anti-6RG antibodies (Figure 55).

6RG::His bound to the Mucin column at a range of pHs, but most strongly at pH4.5, as indicated by the small amount of 6RG::His in the flow-through and the large amount of 6RG::His in the peak eluate fraction. No binding was detected at pH9.0. The overloading of crude extracts to the column resulted in the 6RG::His in the flow through fractions.

Summary of mucin binding results

The carbohydrate-binding properties of the three clones was characterised by analysing the behaviour of the recombinant proteins when passed through a Mucin column at various pHs. In comparison to 4WC and 6RG, the recombinant 7WC appears to have a much narrower pH range over which it is able to bind to mucin (Table 13). Although mucin contains an undefined cocktail of carbohydrates/polysaccharides, differences in the ability of the clones to bind to the column, and differences in the elution profiles at the different pHs indicate: a) the three putative LNPs show lectin- like properties; b) the three clones differ in their carbohydrate-binding profiles (Table 13). pH of 1OmM NaPO₄ buffer

Proteins

3.0 4.5 6.0 7.5 9.0

4WC-M* - +++ +++ +++ +++

4WC-P** +++ +++ +++ +++

6RG::His +++ +++ +++ +++ -

7WC-M* - + +++ +++

7WC-P** - +++ +++ +

7WC::His + +++ +++

(E. col!)

Table 13. Mucin-binding analysis for each protein at different pH buffer

* M represents for the protein from culture medium

** P represents for the protein from lysed cells (cell pellet)

+++ represents strong binding

+ represents poor binding

- represents no binding detected

At pH3.0, 4WC-P isolated from cell pellets was bound to the column, compared to 4WC-M (originating from the culture medium), which did not appear to bind to the column (i.e. no 4WC-M eluted from column). At pH4.5, 4WC-P is predicted to have precipitated as it could not be detected in the crude extract at this pH, while 4WC-M bound to the column at this pH. While the difference in carbohydrate- binding, and a difference in molecular weight suggests that 4WC-M and 4WC-P are potentially differentially cleaved, their amino acid sequences are identical. The difference between 4WC-M and 4WC-P is due to different glycosylation patterns.

Apyrase inhibitors

Levamisole (inhibitor of alkaline phosphatase), sodium fluoride, and ouabain were tested for their

KU potential inhibitory effects on 4WC, 7WC and 6RG apyrase activity. The results were normalised against control reactions containing no inhibitor and are presented in Table 14.

Table 14. Effect of inhibitors on apyrase activity of 4WC, 7WC and 6RG. Apyrase activity has been normalised relative to the control for each clone. The control consisted of the standard apyrsae activity determination protocol in the absence of inhibitor. Apyrase activity such as was calculated from the quantity of free phosphate released by comparing absorbance with inorganic phosphate standards (10, 20, 40, 60, βOnmol PO₄ ^", pH6.8). One unit is defined as 1μmol Pi released by apyrase in one minute per mg protein.

Levamisole and Ouabain appeared to have no effect on any of the apyrases. 4WC was relatively unaffected by NaF, in comparison the apyrase activities of 7WC and 6RG were reduced with increasing concentrations of NaF.

Influence of carbohydrate binding on apyrase activity

Specific carbohydrates have been shown to increase apyrase activity (Etzler et al., 1999; Fonseca et al., 2006). We show here that different carbohydrates interact differently with different apyrases, also that the interaction can result in a decrease or increase of apyrase activity or no change to the level of apyrase activity. Thus the influence of specific carbohydrates on apyrase activity can be used to determine the presence of these carbohydrates via the determination of inorganic phosphate released compared to appropriate controls. Hence, apyrase activity such as was calculated from the quantity of free phosphate released by comparing absorbance with inorganic phosphate standards (10, 20, 40, 60, 80nmol PO₄ ^", pH6.8). One unit is defined as 1μmol Pi released by apyrase in one minute per mg protein.

Specific chitosans (dimer, trimer, tetramer, pentamer and hexamer) were purchased from the Seikagaku Corporation (Japan) and D-galactosamine and D-glucosamine were purchased from Sigma-Aldrich (USA). Apyrase assays were set up as above for 7WC::His, 6RG::His and 4WC::His) using ATP or ADP as the substrate. In addition, either 5μM of a specific chitosan or 6OmM of D- galactosamine or D-glucosamine was added at the same time as the substrate (Table 15). The reactions were incubated for 20-60 minutes and the amount of inorganic phosphate was determined by measuring absorbance at 630nm. For all assays the maximum level of substrate depleted was ≤15% of the starting level. Assays were performed in triplicate; SE and p values were calculated (Table 15).

Table 15. Influence of carbohydrate binding on apyrase activity. Apyrase activity has been normalised relative to the control for each clone. The control consisted of the standard apyrase activity determination protocol in the absence of carbohydrate. Apyrase activity was calculated from the quantity of free phosphate released by comparing absorbance with inorganic phosphate standards (10, 20, 40, 60, δOnmol PO₄ ^", pH6.8). One unit is defined as 1 μmol Pi released by apyrase in one minute per mg protein.

The results from Table 15 shows that we have demonstrated that different carbohydrates either increase, decrease or have no influence on apyrase activity depending on the carbohydrate and apyrase clone. 7WC::His, 4WC::His and 6RG::His interacted differently from each other with different carbohydrates. These differences can be manipulated by swapping the domains thus altering the influence of carbohydrate binding on apyrase activity. 7WC apyrase activity significantly increased in the presence of the chitosan pentamer and hexamer as well as D-galactosmaine and was not significantly influenced by the remaining carbohydrates.

4WC apyrase activity significantly increased in the presence of the chitosan dimer, tetramer and hexmaer as well as D-galactosamine and was not significantly influenced by the remaining carbohydrates. ,;;.

6RG apyrase activity significantly decreased in the presence of chitosan tetramer, pentamer and hexamer as well as D-galactosamine and D-glucosamine. The decrease in apyrase activity is a very important finding as to date there are no reports in the literature of specific carbohydrates negatively influencing apyrase activity. In comparison, 6RG significantly increased apyrase activity in the presence of the chitosan dimer and was not significantly influenced by the remaining carbohydrates.

It should be noted that the influence of chitosan on the activity of the 6RG apyrase can be modified by the type of buffer used where the use of MES (5OmM MES pH6.5, 3mM CaCI2, 5mM ATP) gave an activity of approximately 96 units, the addition of 5mM chitosan hexamer reduced the apyrase activity to approximately 50 units, whereas the use of MOPS (5OmM MOPS pH6.5, 3mM CaCI2, 5mM ATP) gave an activity of approximately 72 units amd this was relatively uninfluenced by the addition of 5mM chitosan hexamer since the activity was approximately 69 units.

Summary of recombinant soluble apyrase mucin binding, substrate preference, pH optimum, inhibitor interaction and influence of carbohydrate binding on apyrase activity

The LNPs can be distinguished from each other based on their different mucin binding properties, substrate specificities, apyrase pH optimum, inhibitory effect of NaF and influence of carbohydrate binding on apyrase activity. The differences are summarised in Table 15. The specific domains responsible for these characteristics have not been identified. However, we demonstrate here that it is possible to manipulate the mucin binding properties, apyrase substrate preference, pH optimum, inhibitor interaction and influence of carbohydrate binding on apyrase activity by fusing different conserved regions from separate clones.

Analysis of pH effect on apyrase activity of 4WC-M, and 6RG::His shows there is a measurable difference in pH optimum (Figures 40, 46 and 49). The apyrase acitivty of 4WC increases with pH and was still increasing at 9.5, in comparison 6RG has a sharp peak of maximum activity at pH 6.5. These differences can be manipulated by swapping the domains; thus altering the pH optimum of the soluble LNPs.

Analysis of substrate preference of 4WC-M, and 6RG::His shows there is a measurable difference in preference (Figures 40, 46 and 49). 4WC demonstrates a moderate preference for NDPs compared to NTPs, in comparison 6RG has a very strong preference for NTPs and has almost no activity with

NDPs. These differences can be manipulated by swapping the domains; thus altering the substrate preference of the soluble LNPs.

Analysis of mucin-binding of 4WC-M, 4WC-P and 6RG::His in 1OmM NaPO₄ Buffer at pH9.0 shows there is a measurable difference in mucin-binding properties between 4WC and 6RG::His(Figures 35, 37, 53, 54 and 55). At pH9.0, recombinant 4WC protein, from either cells or medium, binds to the mucin column while 6RG::His does not. These differences can be manipulated by swapping the domains; thus altering carbohydrate binding properties of the soluble LNPs. Analysis of NaF effect demonstrated that 4WC activity is not inhibited; in comparison the apyrase activity of 6RG is reduced with increasing concentrations of NaF. These differences can be manipulated by swapping the domains thus altering the influence of inhibitors of the soluble LNPs.

We have demonstrated that different carbohydrates either increase, decrease or have no influence on apyrase activity depending on the carbohydrate and apyrase clone. 7WC::His, 4WC::His and 6RG::His interacted differently from each other with different carbohydrates. These differences can be manipulated by swapping the domains thus altering the influence of carbohydrate binding on apyrase activity.

Together the differences between 4WC and 6RG::His in terms of apyrase activity, carbohydrate binding and effect of inhibitors are sufficient to demonstrate the potential to manipulate both charcteristics by swapping selected domains between the soluble apyrases (Table 16).

Table 16. Summary of apyrase activity pH optimum, substrate preference, substrate binding site competition, mucin binding properties, effect of inhibitors, influence of carbohydrate on apyrase activity for 4WC, 7WC and 6RG. Apyrase activity has been normalised relative to the relevant control for each clone. The control consisted of the standard apyrsae activity determination protocol in the absence of inhibitor. Apyrase activity such as was calculated from the quantity of free phosphate released by comparing absorbance with inorganic phosphate standards (10, 20, 40, 60, δOnmol PO₄ ^", pH6.8). One unit is defined as 1 μmol Pi released by apyrase in one minute per mg protein.

Regions of sequence homology

To aid identification of conserved domains, motifs or individual residues, CD39 and a subset of translated peptide sequences from predicted secretory plant apyrases were aligned (Figure 56).

The 4 N-terminal apyrase domains (Handa and Guidotti 1996), the six conserved C-terminal cysteines (Roberts et al., 1999) and the 5th C-terminal apyrase domain (Vasconcelos et al., 1996), were used as primary alignment criteria. CD39, along with the majority of other animal apyrases (not shown) contain an additional two cysteines in their C-terminal half. Figure 56 shows the disulfide bonds as mapped by Ivanenkov et al., (2005). The disulfide bonds can be described as follows: the conserved cysteines in the soluble plant apyrases are consecutively numbered 1 - 6 (with 1 being the residue closest to the N-terminus), there are three disulfide bonds, one between residues 1 and 2, one between residues 3 and 4 and one between residues 5 and 6. In addition to these disulfide bonds the animal apyrases form one extra bond between their additional pair of cysteines which are located on either side of the plant cysteine residue 1. In the plant apyrases, the formation of the three disulfide bonds effectively creates three loops between the respective cysteines and two interloop regions are created between the loops (Figure 56). A similar situation exists in the animal apyrases except that the equivalent of the first loop is intersected by a disulfide bond formed between the extra pair of conserved animal apyrase cysteines. The animal apyrases also contain additional amino acid residues between the corresponding plant cysteine residues 1 and 2; in the case of CD39 there are 6 additional residues.

Other conserved residues that often contribute greatly to a peptides tertiary structure include prolines (which induce a 90° change in the direction of a peptide chain) and asparagine that precede (X)S/T (where X represents any residue except proline while S/T represents either serine or threonine residues). Asparagine residues that precede (X)SfT are potential sites of N-linked glycosylation which often induce a fold. Of the aligned plant apyrases the first, third, fourth and sixth cysteines are generally associated with a close neighbouring proline or potential N-linked glycosylation site or both. Similarly for the corresponding cysteines in animal apyrases such as CD39 (Figure 56). The relatively high conservation and close proximity of such residues indicates that they are likely to be involved in ensuring the formation of the specific disulfide bonds.

While the cysteine residues are absolutely conserved the proline and glycosylation sites are common but not absolutely conserved; as such they are not essential for activity of all apyrases. Furthermore, our successful refolding of active soluble plant apyrases (7WC::His, 6RG::His, 4WC::His, DbLNP::His,

6RG::7WC::His, 7WC::6RG::His, DbLNP::4WC::His, 4WC::DbLNP::His) from bacterial inclusion bodies confirms that N-linked glycosylation is not essential. It should be noted however, in the absence of N-linked glycosylation, the proline induced directional change may be important for efficient correct folding.

An alignment of only five apyrase clones including 4WC, 7WC, 6RG, DbLNP (closely related to 4WC) and CD39 reveals further regions containing conserved proline residues (Figure 57). Notably the first of these is found between the first and second apyrase domain and consists of five proline residues with specific residue spacing between them (i.e. 8, 8, 9 and 7). The second region is a single proline located between the second and third apyrase domains, 4WC does not contain this proline. The last region is immediately after the fourth apyrase domain (prior to the conserved cysteine region) where the plant apyrases contain two prolines with seven residues spaced between them and for two of the plant clones there is an extra proline residue in the space. Instead of two proline residues, CD39 contains one proline and the first of its extra cysteine residues is located very close to this region. Between the second and third ACR the majority of the soluble plant apyrases (and NTPDases) contain a single proline; in addition, a number of the plant apyrases also contain a potential N-glycosylation site 7 residues before this site. The mammalian NTPDases and the soluble plant apyrases contain a proline 6 residues after the fourth ACR and most soluble plant apyrases contain one, two or three additional prolines within a further seven residues.

NTPDases and soluble plant apyrases contain two virtually invariant tryptophans, one is located in the third ARC while the other is located in the fifth ARC. Smith et al., (1999) showed that tryptophan in the third ARC is crucial for activity and likely required for correct folding; in comparison, obliteration of the tryptophan in the fifth ARC resulted in enhanced NTPase activity but reduced NDPase activity. The soluble plant apyrases and mitochondrially targeted plant apyrases contain an additional conserved tryptophan located 5 residues downstream of their predicted second disulfide bond (equivalent to the fourth disulfide bond in the cell surface NTPases). This tryptophan is flanked by a number of highly conserved residues including a string of glycines which could provide a potential hinge region. In comparison, the signal anchored plant apyrases that were not predicted to be mitochondrially targeted ^!- contained a conserved tryptophan 3 residues downstream of their 5th conserved cystenine; similarly for the mammalian cell surface NTPDases, whereas the signal anchored plant apryases that were predicted to be mitochondrially targeted as well as the predicted secretory plant apyrases did not.

Several regions between the C-terminal cysteines are of interest to us; in particular the region containing the potential hinge region which is flanked by the plant disulfide bonds 2 and 3 (4 and 5 in the case of CD39) and the span enclosed by the plant disulfide bond 3 (5 in the case of CD39). We have found that even between very closely related primary sequences these regions often show a relatively large degree of variance in terms of overall residue charge and hydrophobicity. Although the function of these divergent regions is not known, their comparatively high degree of variance suggests there may be sites that distinguish one apyrase from the other. As such, changes in these amino acid sequences likely influence the characteristics of the apyrase.

Secondary structure prediction

JPred (Cuff and Barton, 2000) was used to predict the secondary structures on CD39 and Db-LNP (closely related to 4WC) and NNPREDICT (McClelland and Rumelhart, 1988; Kneller et a/., 1990) was used to predict the secondary structures of CD39, 7WC, 4WC, Db-LNP and 6RG. All clones were predicted to have the virtually same secondary structure (Figure 58).

Two non-polar exposed regions were predicted to occur in the C-terminal half, one of these overlays cysteine loop two and the other overlies the conserved proline rich domain in interloop region two. Exposed hydrophobic stretches were also identified by JPred, these included the first portion of loop one and the first portion of interloop region one. The loop regions appeared to have little in the way of predicted secondary structure. This was limited to two putative short beta sheet regions in the first cysteine loop and a short alpha helix region in the third cysteine loop, predicted by both NNPREDICT (Figure 58) and JPred (not shown).

Inter cysteine loops

Given that we know the location of the disulfide bonds and that only short secondary structures were predicted in these regions, for the moment we have assumed that the rest of the residues form relatively unstructured loops. Between the three loops are two interloop regions and an unflanked tail region follows the third loop (Figures 56-58). If we consider these loops in isolation it is possible to compare their physiochemical properties such as charge and hydrophobicity. The isoelectric points of the three loops and two interloop regions as well as the C-terminal tail were calculated using EMBL (Lehninger (1979) Biochimie (EMBL)). The results are shown in Table 17; this analysis has revealed that there are substantial differences in the charge associated with these regions and between clones. In particular, 7WC has an isoelectric point of greater than 10 in the interloop region two which is adjacent to a region in which the isoelectric point is less than 5 (loop three). The reverse was true of the closely related 4WC clone that has an isoelectric point of 6.5 for interloop region 2 but an isoelectric point greater than 10 for loop 3. In the absence of tertiary information we are limited in terms of interpreting the significance of these regions. However, the amino acid sequences between the conserved cysteines are a comparatively divergent areas within the apyrase family and could be an active site that distinguishes one apyrase from the other.

α°nβ U00P 1 ' r"e5gioJ-n 1P Loop *_ 2 ' r-e*g*ion»* 2 Loop 3 Ta,,

7WC 3.56 10.28 5.5 10.09 4.41 3.67

4WC 3.56 11.42 5.5 6.5 10.18 3.77

6RG 3.68 7.0 6.5 3.83 9.5 4.31

CD39 9.31 5.03 5.5 9.43 4.71 6.18

Table 17. Predicted isoelectric points for the disulfide formed loops and their associated interloop regions.

ExPASy ProtScale hydrophobicity plots Analysis of the hydrophobicity of the C-terminal tail was performed using ExPasy ProtScale with a window size of 7 (Kyte and Doolittle, 1982). The main variance between the plant clones correlated with the variance seen for the predicted isoelectric points, i.e., over interloop region two and loop three (Figure 59).

■ , N-linked glycosylation sites

The conserved C-terminal cysteines are involved in forming three (soluble plant apyrases) or four disulfide bonds (membrane bound animal ectonucleotidases). Formation of the correct disulfide bonds is crucial to attaining the correct form. The position of potential N-linked glycosylation sites relative to other key residues (prolines and cysteines) can reveal their importance in the initial folding procedure. This is especially pertinent in the absence of N-linked glycosylation (as is the case in prokaryotic expression) where the refolding has to be carried out achieved solely by the remaining amino acid residues. There are four relatively conserved N-linked glycosylation sites, the first occurs between the second and third apyrase domains, the second is located five residues upstream of the first plant cysteine, the third is two to four residues upstream of the third plant cysteine, the last is immediately upstream of the fourth plant cysteine. In almost all cases those sequences that are missing any of the first three of these potential N-linked glycosylation sites have a proline in relatively close proximity and a number of the sequences have both, which indicates that they may work in tandem.

Chimeric apyrases to modify pH optimum, substrate specificity, mucing binding properties, effect of apyrase inhibitor and effect of carbohydrate binding on apyrase activity

There is a high degree of sequence conservation and predicted secondary structure within the apyrase family. This suggests they adopt a similar tertiary structure and as such would likely possess similar folding mechanisms. While the C-terminal cysteines are absolutely conserved there is a relatively large degree of variance in terms of residue charge and hydrophobicity within the second intercysteine loop and the third cysteine loop. Although the function of these divergent regions is not known, their comparatively high degree of variance suggests there may be active sites and regions that distinguish one apyrase from the other. As such, changes in these amino acid sequences likely influence the characteristics of the apyrase.

We generated three constructs pCH37 (6RG::4WC), pCH38 (4WC::6RG::His) and pCH43 (6RG::4WC::His) to demonstrate the potential to modify the apyrase activity, pH optimum, substrate specificity, mucin binding properties and effect of apyrase inhibitor on soluble LNPs. While these constructs were made by relatively crude reciprocal grafts between the N-terminal half of one apyrase with the C-terminal half of the other it shows the potential for manipulation (Table 18, Figures 60-62). More specific swapping of individual apyrase domains, individual or paired cysteine loops and inter cysteine loops and or making single point muations in these regions would further refine this potential. Our constructs contained the four N terminal apyrase domains from 1 soluble apyrase and the C- terminal of a second soluble aprase containing the fith apyrase domain and the entire conserved cysteine region. These constructs differ from the chimeras generated by Heine et al., (2001) who chose to use fusions within the 5 apyrase domains and were thus unable to distinguish between the effects of domain swapping with possible effects caused by disruption of the apyrase domains.

Components

Apyrase clone Tag

N-terminal source C-terminal source

pCH37 6RG 4WC none pCH38 4WC 6RG His pCH43 6RG 4WC His

Table 18. Plant apyrase constructs pCH37, pCH38 (4WC::6RG::His) and pCH43 (6RG::4WC::His).

Expression of recombinant apyrase protein (6RG::4WC, 4WC::6RG::His and 6RG::4WC::His) was seen for all three bacmids (pCH37, pCH38 (4WC::6RG::His) and pCH43 (6RG::4WC::His) respectively). All bacmids were taken through to third passage in the insect cells and were of sufficiently high titre for subsequent protein expression studies.

pCH37 did not accumulate in the media; in the cells it accumulated at relatively low levels and appeared to be unstable as seen by the presence of several small (10-30KDa) immunoreactive anti

71 4WC fragments detected by SDS-PAGE/immunoblot (Figure 63). In comparison, pCH38 (4WC::6RG::His) and pCH43 (6RG::4WC::His) were both strongly expressed, pCH38 (4WC::6RG::His) accumulated in both the media and the cell pellet (Figure 64) while pCH43 (6RG::4WC::His) accumulated only in the cell pellet (Figure 65).

pCH38 (4WC::6RG::His) Media Localised Mucin Column Purification _r,

Nickel column purified samples of the media localised pCH38 (4WC::6RG::His) were analysed for their mucin binding properties at 5 different pH values (Figure 66). At each pH tested some pCH38 (4WC::6RG::His) was strongly bound to the mucin column and was eluted with 50OmM NaCI. These characteristics are a blend of both 4WC and 6RG mucin binding characteristics.

pCH38 (4WC::6RG::His) Pellet Localised Mucin Column Purification

Crude pellet extracts were analysed for their pCH38 (4WC::6RG::His) mucin binding properties at 5 different pH values. In contrast to the media localised pCH38 (4WC::6RG::His) the pellet localised protein bound relatively poorly (if at all) to the column at pH 6.0, 7.5 and 9.0, instead it bound and was able to be eluted at pH 4.5 (Figure 66). These characteristics appear to be a blend of both 4WC and 6RG mucin binding characteristics.

pCH38 (4WC::6RG::His) , pCH43 (6RG::4WC::His) apyrase activity, substrate specificity, and pH optimum

The chimera pCH38 (4WC::6RG::His) received 4 N-terminal apyrase domains from 4WC, the conserved cysteine region and the C-terminal 5th apyrase domain from 6RG. pCH38 (4WC::6RG::His) apyrase activity was determined in a range of buffers, including: acetic acid pH3.0, MES pH6.5 and Tris-HCI pH8.5. The substrates were either 5mM ADP or ATP. The apyrase activity (shown as inorganic phosphate released by the apyrase) of each fraction is shown in Figure 67.

pCH38 (4WC::6RG::His) from either the media or pellet had a pH optimum for ATP at ≥9.5 with activity detected at a pH of 3 (Figure 67). The broad pH range is characteristic of 4WC. pCH38 (4WC::6RG::His) from the pellet was able to use both ATP and ADP as substrate with a strong preference for ATP whereas 6RG almost exclusively uses ATP and 4WC uses both ATP and ADP as substrate with a moderate preference for ADP. pCH38 (4WC::6RG::His) from the medium almost exclusively used ATP.

The chimera pCH43 (6RG::4WC::His) receives 4 N-terminal apyrase domains from 6RG, the conserved cysteine region and the C-terminal 5th apyrase domain from 4WC. pCH43 (6RG::4WC::His) accumulated in the cell pellets only and was purified using a NTA column. pCH43 (6RG::4WC::His) had a pH optimum for both ATP and ADP at pH >9.5 (Figure 67). The broad pH range is characteristic of 4WC. pCH43 (6RG::4WC::His) was able to utilise both ATP and ADP with a slight preference for ADP. 4WC in comparison has a moderate preference for ADP.

Effect of NaF on pCH38 (4WC::6RG::His) and pCH43 (6RG::4WC::His) apyrase activity

4WC is unaffected by NaF at either 5 or 1OmM, under the same conditions the apyrase activity of 6RG is reduced. We compared the effect of 1OmM NaF on 6RG; 4WC::His (from the cell and the media); pCH38 (4WC::6RG::His) (from the cell and the media) and pCH43. The results are shown in Table 19.

Table 19. Effect of inhibitors on apyrase activity of 4WC, 6RG, pCH38 (4WC::6RG::His) and pCH43. Apyrase activity has been normalised relative to the control for each clone. The control consisted of the standard apyrase activity determination protocol in the absence of inhibitor. Apyrase activity was calculated from the quantity of free phosphate released by comparing absorbance with inorganic phosphate standards (10, 20, 40, 60, δOnmol PO₄ ^", pH6.8). One unit is defined as 1 μmol Pi released by apyrase in one minute per mg protein.

NaF significantly reduced the apyrase activity of 6RG but had no significant effect on reducing ATP or ADPase activity of 4WC, pCH38 (4WC::6RG::His) or pCH43.

Influence of carbohydrate binding on pCH38 (4WC::6RG::His) , pCH43 (6RG::4WC::His) apyrase activity

Specific chitosans (dimer, trimer, tetramer, pentamer and hexamer) were purchased from the Seikagaku Corporation (Japan) and D-galactosamine and D-glucosamine were purchased from Sigma-Aldrich (USA). Apyrase assays were set up as above for 6RG::His, 4WC::His, pCH38 (4WC::6RG::His) and pCH43 (6RG::4WC::His) using ATP or ADP as the substrate. In addition, either 5μM of a specific chitosan or 6OmM of D-galactosamine or D-glucosamine was added at the same time as the substrate (Table 20). The reactions were incubated for 20-60 minutes and the amount of inorganic phosphate was determined by measuring absorbance at 630nm. For all assays the maximum level of substrate depleted was ≤15% of the starting level. Assays were performed in triplicate; SE and p values were calculated (Table 20).

Table 20. Influence of carbohydrate binding on pCH38 (4WC::6RG::His) and pCH43 (6RG::4WC::His) apyrase activity. Apyrase activity has been normalised relative to the control for each clone. The control consisted of the standard apyrase activity determination protocol in the absence of carbohydrate. Apyrase activity was calculated from the quantity of free phosphate released by comparing absorbance with inorganic phosphate standards (10, 20, 40, 60, 80nmol PO₄ ^", pH6.8). One unit is defined as 1μmol Pi released by apyrase in one minute per mg protein.

Analysis of the influence of carbohydrate binding on apyrase activity 4WC::His and 6RG::His shows that each clone has a different carbohydrate binding preference and that the influence the carbohydrate binding has on apyrase activity (i.e., no effect, increase or decrease in activity) was also different for each clone. 4WC has significantly greater apyrase activity in the presence of the chitosan dimer, tetramer and hexamer as well as D-galactosamine but no effect was seen with the chitosan trimer or pentamer and D-glucosamine. In comparison, 6RG had significantly greater apyrase activity in the presence of the chitosan dimmer but significantly less apyrase activity in the presence of the chitosan tetramer, pentamer and hexamer as well as D-glucosamine and D-glucosamine, whereas the chitosan trimer appeared to have no effect. pCH38 (4WC::6RG::His) showed significant increases in apyrase activity in the presence of the chitosan hexamer and D-galactosmaine, all other sugars had no significant effect on apyrase activity. pCH43 (6RG::4WC::His) showed significantly reduced apyrase activity (both with ATP and ADP as substrate) in the presence of the chitosan pentamer, all other sugars had no significant effect on apyrase activity.

Overall comparison of chimeras

The following is a brief description of the characteristics of 4WC, 6RG, pCH38 (4WC::6RG::His) and pCH43 (6RG::4WC::His).

Subcellular Localisation

Recombinant 4WC was consistently localised in both the cell pellet as well as the media. Below we demonstrate that 4WC inside the cell was likely glycosylated at all 4 potential sites whereas 4WC in the media was probably only glycosylated at 2 sites. Glycosylation at the site N313 was found to be crucial for protein secretion. In comparison, 6RG was consistently localised in the cell pellet only and was glycosylated at all sites. pCH38 (4WC::6RG::His) was localised in both the cell pellet and media whereas pCH43 (6RG::4WC::His) was localised in the cell pellet only. These results would suggest that the signal sequence is also critical since the chimera pCH38 (4WC::6RG::His) was found in both the media and cell pellet whereas the chimera pCH43 (6RG::4WC::His) was localised to the cell pellet.

pH

Analysis of pH effect on apyrase activity of 4WC::His, and 6RG::His shows there is a measurable difference in pH optimum. The apyrase acitivty of 4WC increased with pH and was still increasing at 9.5, in comparison 6RG has a peak of maximum activity at pH 6.5. Both pCH38 (4WC::6RG::His) and pCH43 (6RG::4WC::His) had the same pH optimum curve as seen for 4WC.

Substrate Preference

Analysis of substrate preference of 4WC::His (from the media), and 6RG::His shows there is a measurable difference in preference. 4WC demonstrates a moderate preference for NDPs compared to NTPs (ATP:ADP ~ 2:5). In comparison, 6RG has a very strong preference for NTPs and has almost no activity with NDPs (ATP:ADP ~ 100:1); similarly for pCH38 (6RG::4WC::His) in the media (ATP:ADP -100:1). Whereas pCH38 in the pellet still has a strong preference for NTPs but to a much lesser extent (ATP:ADP -20:1). pCH43 (6RG::4WC::His) had an even more modest preference for NDPs compared to NTPs (ATP:ADP -5:7) than 4WC.

Mucin Binding

Analysis of mucin-binding of 4WC::His (from the media), 4WC::His (from the pellet) and 6RG::His in 1OmM NaPO₄ buffer at pH9.0 shows there is a measurable difference in mucin-binding properties between 4WC and 6RG. At pH9.0, recombinant 4WC protein, from either cells or medium, binds to the mucin column while 6RG does not. pCH38 (4WC::6RG::His) from the media had a similar mucin binding profile to 4WC.

NaF Inhibitory Effect

Analysis of NaF effect demonstrated that 4WC apyrase activity is not inhibited; in comparison the apyrase activity of 6RG was significantly reduced with increasing concentrations of NaF. Apyrase activity of pCH38 (4WC::6RG::His) either from the media or from the cell pellet was not inhibitied by NaF. pCH43 (6RG::4WC::His) ATPase activity was unaffected by NaF, however, the ADPase activity was partially inhibited (although not significantly).

Influence of Carbohydrate Binding on Apyrase Activity

The results are summarised in Table 21.

Table 21. Recombinant protein location, pH optimum, ATP:ADP substrate specificity, mucin binding properties effect of NaF on apyrase activity and effect of carbohydrates on apyrase activity of 6RG::His, 6RG::4WC::His (pCH43), 4WC::6RG::His (pCH38) and 4WC::His (pMW3). Apyrase activity has been normalised relative to the control for each clone. The control consisted of the standard apyrase activity determination protocol in the absence of NaF or carbohydrate. Apyrase activity was calculated from the quantity of free phosphate released by comparing absorbance with inorganic phosphate standards (10, 20, 40, 60, 80nmol PO₄ ^', pH6.8). One unit is defined as 1μmol Pi released by apyrase in one minute per mg protein.

The results of the two chimeras, pCH38 (4WC::6RG::His) and pCH43, indicates that substrate specificity, pH optimum, interaction with inhibitors, mucin binding properties, subcellular localisation and influence of carbohydrate on apyrase activity are not determined by one specific feature such as the N-terminal apyrase domains, the C-terminal 5th apyrase domain, the conserved cysteine region, signal sequence or glycosylation sites, but rather the combination of these features is responsible for determining the catalytic properties of the enzyme. We have therefore demonstrated that it is possible to tailor the apyrase, mucin and carbohydrate binding properties by combining whole regions from independent apyrases. We have also demonstrated the potential to manipulate specific apyrases to recognise and respond to specific carbohydrates which in turn can be used to determine the presence of specific carbohydrates via the colourmetric determination of apyrase activity via inorganic ortho phosphate released compared to appropriate controls.

MODIFICATION OF RECOMBINANT SOLUBLE APYRASE LOCALISATION IN INSECT CELL EXPRESSION

We have demonstrated above that insect cells express active recombinant apyrases and that the recombinant protein can be localised in the cell (6RG) or it can be secreted into the media (4WC). We have also shown that the cell localised recombinant protein has different mucin binding properties to the media localised protein (4WC) and in one case the substrate specificity of the cell localised protein is different to the media localised protein (pCH38 = 4WC::6RG::His). We demonstrate here that it is possible to control the secretion and heterogeneity of the recombinant apyrase by modification of the N-linked glycosylation patterns.

Analysis of N-linked glycosylation

Baculoviral expression of individual full length recombinant soluble apyrase clones can lead to the production of two major isoforms of the encoded protein. The isoforms have the same amino acid sequences but migrate at different rates in SDS-PAGE. They also have different subcellular localisations as well as different mucin binding properties. The larger sized isoform is found exclusively within the cell pellet while the smaller isoform is found in both the cell pellet and the media. We used PNGase F (NEB) to deglycosylate the isoforms and the effect of deglycosylation on electrophoretic mobility was analysed. Following deglycosylation the isoforms had the same electrophoretic mobility indicating their differences is caused by differential glycosylation.

Deglycosylation was carried out using PNGase F (NEB) according to the manufacturers protocols. N-Glycosidase F (from Chryseobacterium [Flavobacterium] menigosepticum), also known as PNGase F, is an amidase that cleaves between the innermost GIcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins.

PNGase F hydrolyzes nearly all types of N-glycan chains from glycopeptides/proteins. However, PNGase F will not cleave N-linked glycans containing core α1-3 Fucose which are commonly found in plant glycosylated proteins. While the apyrases used in this manipulation were originally cloned from plants they will not contain α1-3 Fucose glycosylation motifs since they are being expressed in an insect expression system.

Two reaction conditions, denaturing and non denaturing, were compared when deglycosylating each protein

Deglycosylation, under denaturing conditions was carried out by denaturing the protein in 1 x Glycoprotein Denaturing Buffer (NEB) at 100⁰C 10min, allowed to cool to room temperature and followed by the addition of 1 % NP-40 (this non-ionic detergent counteracts the SDS inhibition of PNGase F activity), 1x G7 Reaction buffer (50 mM NaPO4, pH 7.5) and 1 μL PNGase F.

Deglycosylation under non denaturing conditions was carried out by omitting the denaturation step (Glycoprotein Denaturing Buffer at 100⁰C 10min) and addition of 1 % NP-40. The deglycosylation of native and denatured proteins was carried out on the bench at 22⁰C for varying periods of time.

For each reaction, approximately 20μg of purified or semi-purified LNP was deglycosylated using 1 μL of PNGase F (one unit is defined as the amount of enzyme required to remove >95% of the carbohydrate from 10μg of denatured RNase B in a total reaction volume of 10μL in 1h at 37°C).

Reactions were stopped at various time intervals by taking aliquots and transferring these to protein gel sample loading buffer and boiling for 10 minutes. Samples were then analysed by SDS- PAGE/immunoblot for 4WC and SDS-PAGE/immunoblot and silver stain for 6RG.

4WC from the media Insect expressed recombinant full length 4WC is localised in both the cell pellet and in the media. The cell pellet contains two isomers of 4WC where the slower (larger) migrating isomer accumulates in greater amounts. The media contains only one isomer; this has the same electrophoretic migration properties as the smaller isomer seen in the cell pellet. We have speculated that the smaller isomer detected in the cell pellet is in the process of secretion and has the same glycosylation pattern as the recombinant protein found in the media. Recombinant 4WC for deglycosylation was purified from the media using the protocol outlined in Table 21.

Infect insect cells with relevant construct, grow for 4 days i Remove cells by centrifugation (2,000xg, 5min)

I

Precipitate media using 60-80% ammonium sulphate cut at 4⁰C, centrifuge (39,000xg, 30 min); Resuspend precipitate in Extract Buffer (5OmM Tris HCI, pHδ.0, 10OmM NaCI and 10% glycerol); Dialyse against 10Mm NaPO₄ pH9.0 containing 10% glycerol; Clear debris by centrifugation

(39,000^χg, 30min)

4-

Load in binding buffer onto a mucin column pre-equilibrated with equilibration buffer, 1OmM NaPO₄, pH9.0, 10% glycerol (PsDL6) i

4, Dialyse active⁵ eluted fractions against 5OmM Tris-HCI pH8.0 containing 10% glycerol i

Load onto Ion Exchange column equilibrated with Load Buffer (25mM Tris-HCI pH7.5, 10% glycerol, 20μM PA and 4mM DTT); Wash column with Load Buffer; Elute column with Elution Buffer (Load Buffer + 1 M NaCI), collect 1 mL aliquots for each fraction i Pool and concentrate active fractions by Am icon 10 column

Deglycosylate using NEB PNGase F

I

Collect fractions at various time intervals; Inactivate PNGase F by incubating samples with 25% volume of 4xDenaturing/Reducing Protein Sample Buffer at 100⁰C for 3min

4^, Resolve samples SDS-PAGE i Detect by immunoblot using anti 6RG polyclonal antisera

Table 22. Flow chart to purify and deglycosylate media located full length 4WC.

Denatured 4WC from the media was rapidly (less than two minutes) deglycosylated, this was seen by a shift in protein mobility on SDS-PAGE from approximately 5OkDa to approximately 45.5kDa (Figure 68). Hence, for 4WC expressed in insect cells and secreted into the media the carbohydrate motifs constitute approximately 9% of the total mass of the expressed protein. Longer reaction times did not result in additional mobility; furthermore there were no intermediate protein mobilities present in any of reactions. This would indicate that the denatured protein was fully deglycosylated within two minutes.

Non denatured 4WC from the media also appeared to be fully deglycosylated within two minutes (Figure 69). This rapid rate of deglycosylation of the native 4WC could only occur if the glycosylation motifs were easily accessible to the PNGase F. Such access would indicate that either the motifs are located on the outside of a folded protein or that the location of the glycosylation motifs is in a relatively open space of the protein. After approximately two minutes there was only one anti-4WC immunoreactive band and this migrated to the same position as the deglycosylated denatured 4WC. In order to determine if 4WC from the media has multiple potential glycosylation sites actually glycosylated it would be necessary to dilute the concentration of PNGase F in order to slow down the reaction. It is possible that the rate of deglycosylation reflects the difference between recombinant apyrase secreted into the media compared to the recombinant apyrase retained within the cell.

6RG::His from cell pellets

Mature 6RG has 4 potential glycosylation sites S/T X N, (where X is any amino acid except proline). Recombinant full length 6RG::6His is predominantly localised in the cell pellet and contains two isomers where the larger/slower migrating isomer accumulates in greater amounts. Recombinant 6RG::His for deglycosylation was purified from the cell pellets using the protocol outlined in Table 22.

Infect insect cells with relevant construct, grow for 4 days

I Harvest by centrifugation (2000xg, 5min) i

Wash pellet twice with IxPBS and resuspend in extraction buffer (10OmM NaPO₄ Buffer, pH7.5, 50OmM NaCI, 1 mM PMSF, 1% Nonodet P-40)

I

1

I Deglycosylate using NEB PNGase F

I

, . J,

Resolve samples SDS-PAGE

Detect by immunoblot using anti 6RG polyclonal antisera

Table 23. Flow chart to purify and deglycosylate cell located full length 6RG::6His

Denatured 6RG::His was rapidly (less than 2min) deglycosylated, this was seen by a shift in protein mobility on SDS-PAGE from approximately 58KDa to approximately 48KDa (Figure 70). Hence, for 6RG::His expressed in insect cells the carbohydrate motifs constitute approximately 17% of the total mass of the expressed protein. Longer reaction times did not result in additional mobility, furthermore there were no intermediate protein mobilities present in any of reactions. This would indicate that the denatured protein was fully deglycosylated within two minutes.

In comparison, non denatured 6RG::His appeared to be deglycosylated in multiple stages as seen by the presence of multiple distinctive bands where fully glycosylated protein has the slowest electrophoretic mobility (0 minute lane) and the fully deglycosylated protein (10 minute lane) has the fastest electrophoretic mobility (Figure 71). Given that PNGase F cleaves each glycosylation motif only once, the results suggest that at least two and possibly three of the proteins four potential glycosylation sites are indeed glycosylated. The fact that the intermediate bands are as sharply focussed as the fully deglycosylated protein indicates that the glycosylation motifs for each site are approximately the same size (2-3KDa). Furthermore, the fact that all bands showing deglycosylated 6RG were more sharply focussed than the fully glycosylated protein indicates that there is considerable glycosylation heterogenity of the recombinant protein.

The rate of deglycosylation of the native 6RG::His from the cell pellet was also relatively rapid (approximately 10min to virtually complete); this would only occur if the glycosylation motifs were easily accessible to the PNGase F. Such access would indicate that either the motifs are located on the outside of a folded protein or that the location of the glycosylation motifs is in a relatively open space of the protein. After approximately 10min the majority of the native 6RG::His from the cell pellet had been deglycosylated with a lesser amount of protein remaining with a single glycosylation motif still attached.

Site selection for mutational analysis of N-linked glycosylation on 4WC

In the majority of our insect cell expression batches recombinant 4WC was found in both the media and the cell. SDS-PAGE analysis of glycosylated and deglycosylated media localised 4WC showed that N-linked glycosyl motifs constituted approximately 5-5.5KDa. In comparison, the same analysis revealed that N-linked glycosyl mofifs constituted approximately 10KDa on cell-localised 6RG (roughly double the amount found on media localised 4WC). We were able to use limiting amounts of PNGase F to demonstrate that cell localised 6RG is glycosylated at each of its four potential sites by uniformly sized glycosylation motifs (Figure 71). We estimate that each motif constitutes approximately 2.5KDa, i.e., roughly 14 six-carbon sugar residues (this is also the same size as the initial glycosylation decoration common to eukaryotes). If we assume that the insect cell expression system uses relatively uniform glycosylation motifs then media-localised 4WC is likely to be glycosylated at only two of its potential four sites whereas the slower migrating cell-localised 4WC is likely to be glycosylated at 3 or all of its potential sites. The dual localisation of 4WC and their different glycosylation states indicates the importance of glycosylation and pattern of glycosylation on the localisation of the recombinant apyrase in insect cells. N-linked glycosylation requires the presence of a suitable secretory signal to allow the forming protein to enter the endoplasmic reticulum. From here there are many sites that the protein can be targeted too; this is dependent on a number of factors including the signal sequence itself as well as the position of potential N-linked glycosylation sites. The virtual absence of recombinant 6RG in the media could be due to either the absence of a suitable signal sequence and or the lack of appropriate N-linked glycosylation sites.

The alignment in Figure 56 shows the position of the potenital glycosylation sites relative to the postion of the apyrase domains, conserved cysteine residues and semi conserved proline residues. Generally, aprases contain four relatively conserved N-linked glycosylation sites, the first occurs between the second and third apyrase domains, the second is located five residues upstream of the first cysteine. The third and fourth sites are the most highly conserved and the majority of apyrases have one or both sites present. The third site is. 2-4 residues upstream of the third cysteine and the fourth site is upstream and adjacent to the fourth cysteine. In almost all cases those sequences that are missing any of the first three of these potential N-linked glycosylation sites have a proline in relatively close proximity and a number of the sequences have both. 4WC has four relativley well spaced potential N- linked glycosyation sites whereas 6RG has its four potential sites clustered in two regions, both of which are located in the C-terminal half of the protein (Figure 72). In order to test the importance of the N-terminal N-linked glycosylation sites for subcellular targeting on 4WC in the insect cell expression system we chose to abolish the site closest to the N-terminus (N85) by the mutation N85-A85. The high degree to which apyrases have a conserved potential N-linked glycosylation site that flank the third conserved cysteine also flag the importance of this site (N313); hence it was also also chosen for mutational analysis (N313-A313). The mutations are shown schematically in Figure 72.

Generation ofpCH45 (C-terminal mutation, N313-A313).

The QuikChange® Il Site-Directed Mutagenesis kit (Stratagene) was used in combination with the primer paire shown in Table 22 to mutate asparagine residue 313 to alanine.

Generation ofpCH47 (N-terminal mutation, N85-A85)

The mutation was introduced by PCR using overlapping primers with internal mutations (Table 24).

Generation ofpCH48 (N and C-terminal mutations, N85-A85; N313-A313)

The N-terminus of pCH47 was ligated in frame to the C-terminus of pCH45 after restriction digestion using Bam HI.

Table 25. Primers used to generate glycosylation mutants pCH45 and pCH47. Mutation sites are indicated by bold face and underlining.

Expression of glycosylation mutants

Once the initial cloning of the mutants had been completed and checked, the relevant sequences were transferred into BaculoDirect C-terminal linear DNA and transfected into Sf9 insect cells (as described in PsDL5). After increasing the titre to 3 * 10⁸, 7.5 x 10⁸ and 10 * 10⁸ for pCH45, pCH47 and pCH48, respectively, the lines were cultured and the media and cells harvested after 5 days.

Properties of glycosylation mutants

At 0, 24, 48, 72, 96 and 120 hours post-infection samples were centrifuged to separate the cells from the media remove and analysed by SDS-PAGE/immunoblot using anti-4WC antibodies (Figure 73).

Wild type 4WC and pCH47 (N85-A85) were found in both the cell pellets and in the media. In comparison pCH45 (N313-A313) and pCH48 (N85-A85; N313-A313) were only found in the cell pellets and were not detected in the media. These results demonstrate that the glycosylation site at N313 is essential for media localisation.

Expression/accumulation of the recombinant protein is delayed when either of the glycosylation sites are removed (Figure 73).

Gel shift analysis of mutants

Protein extracts from cell pellets were analysed by SDS-PAGE/immunoblot to determine if the mutations had caused any change in electrophoretic migration ability (Figure 74. The results show that pCH47 (N85-A85) does not appear to have any change in migration mobility compared to wild type where the two isoforms were completely resolved. In comparison, in pCH45 (N313-A313) and the double mutant pCH48(N85-A85; N313-A313) the larger isoform migrated slightly faster than the wild type large isoform such that in both pCH45 and pCH48 the two isoforms were no longer fully resolved on a 10% acrylamide gel. The lack of gel shift for either isoform produced in pCH47 compared with the wild type would indicate that the potential N-linked glycosylation site at N85 on 4WC is likely not glycosylated in the insect cell expression system. This correlates with the lack of N85-A85 mutational influence on protein targeting (Figure 74). In comparison, the N313-A313 mutation in pCH45 and pCH48 abolished media localisation and caused a gel shift for the larger 4WC isoform. We predict that 4WC located in the media is normally glycosylated at only two of its sites in insect cells; the abolition of one of those sites may or may not cause a change in the targeting of the recombinant protein but would most certainly cause a shift in the electrophoretic mobility. These results indicate that in the insect expression system 4WC located in the media is glycosylated at N313 and not at N85. Furthermore, the glycosylation of site N313 is involved in the targeting of the protein to the media.

Thus we have demonstrate that 4WC inside the cell was likely glycosylated at all 4 potential sites whereas 4WC in the media was probably only glycosylated at 2 sites. Glycosylation at the site N313 was found to be crucial for protein secretion. In comparison, 6RG was consistently localised in the cell pellet only and was glycosylated at all sites. pCH38 (4WC::6RG::His) was localised in both the cell pellet and media whereas pCH43 (6RG::4WC::His) was localised in the cell pellet only. These results would suggest that the signal sequence is also critical since the chimera pCH38 (4WC::6RG::His) was found in both the media and cell pellet whereas the chimera pCH43 (6RG::4WC::His) was localised to the cell pellet. Effect ofN-linked glycosylation mutations on pH apyrase activity optimum

We tested both the effect of the mutations on pH optimum since wild type 4WC is an active apyrase over a very broad pH range. We also tested the effect of the mutations on substrate specificity since wild type 4WC has a moderate preference for NDPs versus NTPs.

The effect of the N-A mutations on apyrase activity and pH optimum was determined using the same protocol as described above; the results are shown in Figure 75. Whole cells were used to conduct the assay. As a consequence of the whole cell assays we took two steps to ensure the apyrase activities of each recombinant protein could be compared with each other (absolute apyrase activities cannot not be calculated); these are as follows. • Use the same quantity of recombinant apyrase in each step. Using SDS-

PAGE/immunoblot to determine the amount of recombinant protein in each sample.

• Subracting the background Sf9 cell apyrase activity. All eukaryotes have apyrases; hence it could be expected that untransformed insect cells will contain some apyrase activity. We calculated the amount of ^"recombinant apyrase in each transformed cell line to determine the number of cells to compare with each other. For the Sf9 background determination we used more cells than the highest cell number required for the recombinant expressing cell lines. This was to ensure that we would over estimated rather than underesitmate the background; we found the activity in the Sf9 cells was relatively low.

The glycosylation mutations appeared to have no affect on the apyrase activity of 4WC; similarly they did not appear to affect the pH optimum (Figure 73). This indicates that glycosylation at N85 and or N313 in the insect cell expression system is not essential for apyrase activity.

Effect of N-glycosylation mutants on apyrase substrate preference

The effect of the N-A mutations on apyrase substrate preference was determined using the same protocol as described above. As for determining the effect of the N-A mutations on pH optimum whole cells were used to conduct the assay; similarly we took the same two steps to ensure the recombinant proteins can be compared with each other. The results are shown in Figure 76.

The glycosylation mutations appeared to have no affect the apyrase substrate preference of 4WC. This indicates that glycosylation at N85 and or N313 in the insect cell expression system does not determine apyrase substrate preference for recombinant 4WC.

Effect on Carbohydrate/Mucin binding

Determination of carbohydrate binding properties of soluble recombinant 4WC was carried out as described above. Lysate from harvested cells was passed through a mucin column and fractions eluted, using various buffers covering a wide pH range (pH 3.0 to 9.0 in increments of 1.5 pH units). Fractions collected from the column were initially analysed on SDS-PAGE gels by silver staining then by immunoblot (Figure 77). This showed that all glycosylation mutants bound to the mucin column at pH 6.0 and 7.5. Wild type recombinant protein also binds at these pH values, hence the deletion of one or both glycosylation sites has not affected mucin binding at these pH values.

Manipulation of N-giycosylation sites to generate homogeneous recombinant soluble apyrases

The recombinant 4WC glycosylation mutants pCH45, pCH47 and pCH48 were successfully expressed in the insect cell line Sf9. Abolition of the glycosylation site N85-A85 (pCH47) did not affect targeting and like the wild type 4WC the recombinant pCH47 protein was found both inside the cell as well as in the media. In comparison mutation of the third glycosylation site N313-A313 (pCH45) stopped the protein from being secreted to the media.

Using equal amounts of recombinant protein and subtracting the background Sf9 cell apyrase activity we have shown that the apyrase activity, pH optimum, and substrate preference were unaffected by either the single or the double mutations. Similarly, the mutations did not affect mucin binding at pH 6.0 or 7.5. These results are in contrast to recombinant glycosylation mutants of rat CD39 which had reduced activity and stability when expressed in COS7 cells (Wu et al., 2005). They reported that in general, deglycosylation lead to reduced recombinant protein accumulation and reduced apyrase activity; some sites were more critical than others in their effect and the effects were cumulative. The exception to this was their Δ4 CD39 mutant which is the equivalent to our pCH45 mutant (N313-A313. The Δ4 mutant had a moderate reduction in apyrase activity (possibly caused by reduced expression since the results were not normalised) but a high resistance to cleavage. This mutation would generate a useful trait for manufacturing recombinat soluble apyrase as it would allow the production of a more homogeneous protein (in terms of glycosylation) that is only located in the cells and one that was more robust.

In addition to the presence of specific glycosylation sites, the signal sequence is critical since the hybrid pCH38 (4WC::6RG::His) was found in both the media and cell pellet whereas the 6RG::His and pCH43 (6RG::4WC::His) were localised to the cell pellet.

We have demonstrated here that by the abolition of one specific N-linked glycosylation site (in the region of plant conserved cysteines 3 and 4) it is possible to produce an active and comparatively homogeneously glycosylated soluble apyrase that is not secreted. CHIMERIC APYRASES TO MODIFY SPECIFIC GLYCAN BINDING PROPERTIES

6RG, 4WC, pCH38 (4WC::6RG::His) and pCH43 (6RG::4WC::His) were expressed in insect cells and purified as above. Approximately 20μg of each recombinant protein was incubated on a glycan printed array containing 285 individual glycans covalently linked to the slide (http://www.functionalglycomics.org/static/consortium/organization/sciCores/coreh.shtml). The slides were incubated in either: 5OmM Acetate buffer pH 5.0, 3mM calcium chloride, 0.05% Tween 20, 1 % BSA, 5mM ATP; or 5OmM Tris-HCI pH 7.2, 3mM calcium chloride, 0.05% Tween 20, 1% BSA, 5mM ATP. Following several washes in the appropriate buffer the slides incubated with 6RG were probed with rabbit anti-6RG @ 1/500 while the slides incubated with 4WC, pCH38 or pCH43 were probed with rabbit anti-4WC @ 1/500. Carbohydrate binding was then determined indirectly by Relative Fluorescence Units (RFU) emitted by FITC labelled secondary antibody. Analysis of glycan binding

Numerical manipulation of data.

The data was not normalised the data as this appears to skew the data when the maximum RFU was comparatively low (e.g., 6RG at pH 7.2). Comparison of absolute RFU units between clones is not appropriate since anti 6RG was only used for the 6RG clone and anti-4WC may have a preference for the N-terminus of 4WC compared with the C-terminus (seen by the overall difference between the RUF data for the 4WC::6RG in comparison to the 6RG::4WC chimera). In addition, antibody titres and protein concentrations may have varied between slides.

Removal of anti-apyrase antibody glycan low affinity and high affinity binding background.

To determine the anti apyrase antibody glycan binding background, Core-H ran slides using only Anti-

6RG or Anti-4WC antiserum. Both of these produced a low level of background binding to many glycans; this was subtracted from the actual apyrase RFU. In addition, there were some glycans to which each antibody appeared to bind with high affinity (13, 56, 61 , 62, 81-86, 100, 108-112, 124, 132, 149, 168, 170, 180, 195, 196, 218, 240). Consequently the results from these glycans were removed from all data sets since this reduces the confusion when comparing between apyrases that have used different antibodies for detection.

The 6RG, 4WC, pCH38 (4WC::6RG::His) and pCH43 (6RG::4WC::His) glycan binding data corrected 5 for anti apyrase high affinity and low affinity glycan binding is shown in Table 26.

Glycan Glycan Name RFU RFU RFU RFU RFU RFU RFU R]

No. 6RG 6RG 4WC 4WC 6RG:4WC 6RG:4WC 4WC:6RG 4WC

(high (low (high (low (high pH) (low pH) (high pH) (low pH) pH) pH) pH)

AGP 1356

1 3130 2 7190 4501 903 2851 13610

AGP-A 1021 1029

2 1461 5 0 4769 432 1674 9674

AGP-βl 1105

3 3911 8424 0 5109 186 3109 9081

Ceruloplasmine 1088 1654 2173 1783

4 9 8 3 2 3858 9910 12572

5 Fibrinogen 1192 4418 4647 1932 852 1776 7485

Transferrin 1151 1187

6 9 9751 3 1914 2976 6301 18691

7 α-D-Gal-Sp8 437 431 1731 1125 695 1498 4461

8 α-D-Glc-Sp8 849 1483 846 2475 556 2764 4595

9 α-D-Man-Sp8 842 2500 1339 1830 86 747 1733

10 α-GalNAc-Sρ8 337 687 388 596 125 885 290

11 α-L-Fuc-Sρ8 264 1034 627 6387 619 1673 2705

12 α-L-Fuc-Sρ9 421 176 650 -39 -195 383 5222

13 α-L-Rhα-Sp8 0 0 0 0 0 0 0

14 α-Neu5Ac-Sρ8 1012 2056 2922 -83 -1241 -1099 3178

15 α-Neu5 Ac-Sp 11 1027 3690 3114 2782 597 944 7618

16 β*feu5Ac-Sp8 ^' -27 1299 2406 1661 207 862 1535

17 β-D-Gal-Sp8 1465 792 4389 2687 576 2215 1792

18 β-D-Glc-Sp8 912 1005 3441 1540 634 2000 2933

19 β-D-Man-Sp8 610 478 855 408 -107 326 1107

20 β-GalNAc-Sp8 569 1896 1052 2119 977 1893 1371

21 β-GlcNAc-SpO -135 293 972 745 0 1121 573

22 β-GlcNAc-Sp8 482 595 272 274 0 1077 492

23 β-GlcN(Gc)-Sp8 -77 279 2049 1211 34 240 2023

(Galβl-4GlcNAcβ)₂-3,6-

24 GalNAcα-Sρ8 775 225 1926 267 214 304 2356

GlcNAcβl-

3(GlcNAcβl-

4)(GlcNAcβl-

25 6)GlcNAc-Sp8 8 -6 -461 234 -1007 664 168

[3OSO3][6OSO3]Galβl- 4277 3982 3784 4076

26 4[6OSO3]GlcNAcβ-Sp0 0 4 1 5 11723 31454 50194

[3OSO3][6OSO3]Galβl- 1457 3623 1799 2723

27 4GlcNAcβ-SpO 3 9 6 9 3308 12284 27736

[3OSO3]Galβl-4Glcβ-

28 Sp8 1223 3279 4276 4950 1075 1768 8218 [3OSO3]Galβl- 1049 2334 1831 2494

4(6OSO3)Glcβ-Sp0 1 7 8 1 5956 11500 28238

[3OSO3]Galβl- 1187 1641 2704 2746

4(6OSO3)Glcβ-Sp8 0 5 0 4 4930 11467 31765

[3OSO3]Galβl-3(Fucαl-

4)GlcNAcβ-Sp8 648 2416 2303 1947 -156 742 4503

[3OSO3]Galβl- •

3GalNAcα-Sρ8 836 8796 1501 2534 317 2869 3648

[3OSO3]Galβl-

3GlcNAcβ-Sp8 573 5496 2416 1826 -282 2097 5214

[3OSO3]Galβl-4'(Fucαl-

3)GlcNAcβ-Sp8 428 5608 362 168 -483 560 1880

[3OSO3]Galβl- 1466 2240 2169 3013

4[6OSO3]GlcNAcβ-Sρ8 0 4 2 8 3781 13936 29826

[3OSO3]Galβl-

4GlcNAcβ-SpO 711 6298 2255 2029 -231 594 3299

[3OSO3]Galβl-

4GlcNAcβ-Sρ8 1033 7573 2189 3286 749 1794 5183

[3OSO3]Galβ-Sp8 1097

3094 4397 9 7668 1473 3804 13221

[4OSO3][6OSO3]Galβl- 2660 4190 2518 3621

4GlcNAcβ-SpO 6 8 4 1 5598 19632 30454

[4OSO3]Galβl-

4GlcNAcβ-Sρ8 601 6131 2931 3360 955 1591 7128

6-H₂PO₃Manα-Sp8 1634

2280 6344 7 9958 4176 6839 12540

[6OSO3]Galβl-4Glcβ-

SpO 1350 4491 5842 4165 2261 4036 11648

[6OSO3]Galβl-4Glcβ-

Sp8 969 4310 3580 2927 1316 2515 11707

[6OSO3]Galβl-

4GlcNAcβ-Sp8 1712 6698 7236 5559 1356 2682 8182

[6OSO3]Galβl- 1089 1829 1971 2331

4[6OSO3]Glcβ-Sp8 5 1 0 0 5263 12483 25274

NeuAcα2-

3[6OSO3]Galβl-

4GlcNAcβ-Sp8 847 7189 4565 6248 392 1590 8450

[6OSO3]GlcNAcβ-Sp8 1541 6292 -3576 -3576 -3576 -3576 -3576

9-O-AcNeu5NAcα-Sρ8 452 1260 1660 1182 170 1078 2649

9-O-AcNeu5NAcα2-

6Galβl-4GlcNAcβ-Sp8 115 375 2539 1234 23 -46 1135

Manαl-3(Manαl-

6)Manβl-4GlcNAcβl-

4GlcNAcβ-Gly 219 984 1293 727 394 111 3920

GlcNAcβl-2Manαl-

3(GlcNAcβl-2Manαl-

6)Manβl-4GlcNAcβl-

4GlcNAcβ-Gly 1063 1644 910 880 540 141 2428

Galβl-4GlcNAcβl-

2Manαl-3(Galβl-

4GlcNAcβl-2Manαl-

6)Manβl-4GlcNAcβl-

4GlcNAcβ-Gly 129 434 1697 246 -32 96 2423

Neu5Acα2-6Galβl-

4GlcNAcβl-2Manαl-

3(Neu5Acα2-6Galβl-

4GlcNAcβl-2Manαl-

6)Manβl-4GlcNAcβl-

4GlcNAcβ-Gly 147 644 540 -144 -39 166 1773 Neu5Acα2-6Galβl-

4GlcNAcβl-2Manαl-

3(Neu5Acα2-6Galβl-

4GlcNAcβl-2Manαl-

6)Manβl-4GlcNAcβl-

4GlcNAcβ-Sp8 8 393 1300 22 -18 200 2740

Fucαl-2Galβl-

3GalNAcβl-3Galα-Sp9 299 230 426 74 307 227 1577

Fucαl-2Galβl-

3GalNAcβl-3Galαl-

4Galβl-4Gle'β-Sp9 0 0 381 328 -30 205 2082

Fucαl-2Galβl-3(Fucαl-

4)GlcNAcβ-Sp8 522 3348 174 65 19 32 1108

Fucαl-2Galβl-

3GalNAcα-Sρ8 -71 -93 121 -240 -318 -156 244

Fucαl-2Galβl-

3GalNAcβl-

4(Neu5Acα2-3)Galβl-

4Glcβ-SpO 210 625 447 381 -106 647 1997

Fucαl-2Galβl-

3GalNAcβl-

4(Neu5Acα2-3)Galβl-

4Glcβ-Sp9 501 39 491 529 -290 327 1357

Fucαl-2Galβl-

3GlcNAcβl-3Galβl-

4Glcβ-SplO 0 0 0 0 0 0 0

Fucαl-2Galβl-

3GlcNAcβl-3Galβl-

4Glcβ-Sp8 0 0 0 0 0 0 0

Fucαl-2Galβl-

3GlcNAcβ-Sp0 -172 -33 All 232 6 172 2898

Fucαl-2Galβl-

3GlcNAcβ-Sp8 33 555 -47 -29 -63 414 2309

Fucαl-2Galβl-4(Fucαl-

3)GlcNAcβl-3Galβl-

4(Fucαl-3)GlcNAcβ-

SpO 105 233 639 525 388 247 2112

Fucαl-2Galβl-4(Fucαl-

3)GlcNAcβl-3Galβl-

4(Fucαl-3)GlcNAcβl-

3Galβl-4(Fucαl-

3)GlcNAcβ-SρO -14 144 190 43 -505 67 383

Fucαl-2Galβl-4(Fucαl-

3)GlcNAcβ-Sp0 626 340 1936 511 -206 -200 2784

Fucαl-2Galβl-4(Fucαl-

3)GlcNAcβ-Sp8 502 179 75 -62 -45 -113 2775

Fucαl-2Galβl-

4GlcNAcβl-3Galβl-

4GIcNAc-SpO 8 636 45 -73 49 74 2006

Fucαl-2Galβl-

4GlcNAcβl-3Galβl-

4GlcNAcβ-SpO -20 121 110 256 -141 454 392

Fucαl-2Galβl- ^•

4GlcNAcβ-SpO 1501 502 1041 1011 307 660 2959

Fucαl-2Galβl-

4GlcNAcβ-Sp8 -7 -131 1350 768 288 642 2741

Fucαl-2Galβl-4Glcβ-

SpO 297 794 2011 1584 389 757 3679

Fucαl-2Galβ-Sp8 374 -38 1406 715 -467 831 2380 75 Fucαl-3GlcNAcβ-Sp8 214 304 1168 711 477 1828 3703

16 Fucαl-3GlcNAcβ-Sp8 -246 54 44 490 -173 680 234

11 Fucαl-4GlcNAcβ-Sp8 73 135 639 -25 -149 135 455

78 Fucβl-3GlcNAcβ-Sp8 55 298 498 638 458 1070 2300

GalNAcαl-3(Fucαl-

19 2)Galβl-3GlcNAcβ-SpO -126 -34 617 775 -1052 -602 -235

GalNAcαl-3(Fucαl-

2)Galβl-4(Fucαl-

80 3)GlcNAcβ-SpO -286 -10 -69 16 -521 318 751

GalNAcαl-3(Fucαl-

81 2)Galβfi4GlcNAcβ-SpO 0 0 0 0 0 0 0

GalNAcαl-3(Fucαl-

82 2)Galβl-4GlcNAcβ-Sp8 0 0 0 0 0 0 0

GalNAcαl-3(Fucαl-

83 2)Galβl-4Glcβ-SpO 0 0 0 0 0 0 0

GalNAcαl-3(Fucαl-

84 2)Galβ-Sp8 0 0 0 0 0 0 0

GalNAcαl-3GalNAcβ-

85 Sp8 0 0 0 0 0 0 0

86 GalNAcαl-3Galβ-Sp8 0 0 0 0 0 0 0

GalNAcαl-4(Fucαl-

87 2)Galβl-4GlcNAcβ-Sp8 200 1473 98 694 -134 752 429

GalNAcβl-3GalNAca-

88 Sp8 -188 221 -198 647 -313 1497 484

GalNAcβl-3(Fucαl-

89 2)Galβ-Sp8 688 2153 159 810 89 746 332

GalNAcβl-3Galαl-

90 4Galβl-4GlcNAcβ-SpO -150 -277 158 603 -203 126 486

GalNAcβl-4(Fucαl-

91 3)GlcNAcβ-SpO -52 2083 459 1320 -446 642 2412

GalNAcβl-4GlcNAcβ-

92 SpO 133 240 514 672 -312 -82 603

GalNAcβl-4GlcNAcβ-

93 Sp8 -103 -163 631 1361 504 2575 533

94 Galαl-2Galβ-Sp8 -261 477 1464 141 -804 -418 1010

Galαl-3(Fucαl-2)Galβl-

95 3GlcNAcβ-Sρ0 275 1376 61 7 199 474 2059

Galαl-3(Fucαl-2)Galβl-

4(Fucαl-3)GlcNAcβ-

96 SpO 715 719 12 -186 324 825 4017

Galαl-3(Fucαl-2)Galβl-

97 4GIcNAc-SpO 334 380 -263 -2 -267 -92 2061

Galαl-3(Fucαl-2)Galβl- •

98 4Glcβ-Sp0 270 1972 1400 -236 -298 412 4200

Galαl-3(Fucαl-2)Galβ-

99 Sρ8 448 762 95 337 -239 -53 1808

Galαl-3(Galαl-4)Galβl-

100 4GlcNAcβ-Sρ8 0 0 615 152 -78 993 1778

101 Galαl-3GalNAcα-Sρ8 -161 -14 -66 623 69 1119 1518

102 Galαl-3GalNAcβ-Sp8 207 1163 548 1567 106 967 1080

Galαl-3Galβl-4(Fucαl-

103 3)GlcNAcβ-Sp8 279 364 10 238 128 955 3909

Galαl-3Galβl-

104 3GlcNAcβ-SpO 377 267 111 100 349 271 3254

Galαl-3Galβl-

105 4GlcNAcβ-Sρ8 15 106 -658 -500 -279 133 2835

Galαl-3Galβl-4Glcβ-

106 SpO 712 334 3282 1523 555 1031 4766 107 Galαl-3Galβ-Sp8 302 1798 1188 1272 431 774 2768

Galαl-4(Fucαl-2)Galβl-

108 4GlcNAcβ-Sp8 0 0 0 0 0 0 0

Galαl-4Galβl-

109 4GlcNAcβ-SpO 0 0 0 0 0 0 0

Galαl-4Galβl-

110 4GlcNAcβ-Sp8 0 0 0 0 0 0 0

Galαl-4Galβl-4Glcβ-

111 SpO 0 0 0 0 0 0 0

112 Galαl-4GlcNAcβ-Sρ8 254 188 932 909 -52 1446 2119

113 Gaϊαl-6Glcβ-Sp8 -1844 961 3785 1976 -377 556 1712

114 Galβl-2Galβ-Sp8 286 1380 1007 314 -148 1321 1143

Galβl-3(Fucαl-

4)GlcNAcβl-3Galβl-

4(Fucαl-3)GlcNAcβ-

115 SpO 396 984 197 137 223 267 2473

Galβl-3(Fucαl-

4)GlcNAcβl-3Galβl-

116 4GlcNAcβ-Sp0 227 1526 667 220 -21 170 990

Galβl-3(Fucαl-

117 4)GlcNAc-SpO 281 609 498 410 -14 . 218 2521

Galβl-3(Fucαl-

118 4)GlcNAc-Sp8 -132 1251 518 355 -44 746 1502

Galβl-3(Fucαl-

119 4)GlcNAcβ-Sp8 350 352 514 704 194 155 600

Galβl-3(Galβl-

4GlcNAcβl-

120 6)GalNAcα-Sp8 20 698 267 889 -152 297 2009

Galβl-3(GlcNAcβl-

121 6)GalNAcα-Sρ8 -218 301 3 1391 -761 -227 949

Galβl-3(Neu5Acα2-

122 6)GalNAcα-Sρ8 421 879 716 1886 23 1236 1290

Galβl-3(Neu5Acβ2-

123 6)GalNAcα-Sp8 101 893 463 797 382 1118 4888

Galβl-3(Neu5Acα2-

6)GlcNAcβl-4Galβl-

124 4Glcβ-SplO 0 0 0 0 -674 0 0

125 Galβl-3GalNAcα-Sρ8 -48 1167 832 1092 685 370 1777

126 Galβl-3GalNAcβ-Sp8 251 563 809 1588 -189 -8 2147

Galβl-3GalNAcβl-

3Galαl-4Galβl-4Glcβ-

127 SpO 217 577 371 1149 1037 235 2183

Galβl-3GalNAcβl-

4(Neu5Acα2-3)Galβl-

128 4Glcβ-SpO 452 275 217 420 -140 242 2162

Galβl-3GalNAcβl-

129 4Galβl-4Glcβ-Sp8 45 524 586 1506 231 872 50

130 Galβl-3Galβ-Sp8 -75 765 1225 2304 486 1652 3044

Galβl-3GlcNAcβl-

131 3Galβl-4GlcNAcβ-SpO 68 1032 355 -376 -314 306 -95

Galβl-3GlcNAcβl-

132 3Galβl-4Glcβ-SplO 0 0 0 0 0 0 0

133 Galβl-3GlcNAcβ-Sρ0 212 485 864 853 355 2438 2977

134 Galβl-3GlcNAcβ-Sp8 -52 1156 640 977 281 835 3430

Galβl-4(Fucαl-

135 3)GlcNAcβ-Sp0 -1 339 1412 1095 424 467 3455

Galβl-4(Fucαl-

136 3)GlcNAcβ-Sp8 -27 1147 574 551 275 529 2776 Galβl-4(Fucαl-

3)GlcNAcβl-4Galβl-

4(Fucαl-3)GlcNAcβ-

137 SpO 131 241 723 937 -629 1136 919

Galβl-4(Fucαl-

3)GlcNAcβl-4Galβl-

4(Fucαl-3)GlcNAcβl-

4Galβl-4(Fucαl-

138 3)GlcNAcβ-SpO 89 1135 1236 -408 -792 -672 1853

Galβl-4[6OSO3]Glcβ-

139 TfSpO 1093 3960 2719 2240 309 2353 6789

Galβl-4[6OSO3]Glcβ-

140 Sp8 528 1360 2838 4558 639 638 8973

Galβl-4GalNAcαl-

3(Fucαl-2)Galβl-

141 4GlcNAcβ-Sp8 4 214 307 1177 -701 671 1461

Galβl-4GalNAcβl-

3(Fucαl-2)Galβl-

142 4GlcNAcβ-Sp8 -173 521 195 864 33 230 2458

Galβl-4GlcNAcβl-

3(Galβl-4GlcNAcβl-

143 6)GalNAcα-Sp8 85 46 27 93 -184 78 494

Galβl-4GlcNAcβl-

144 3GalNAcα-Sp8 170 479 158 652 -174 55 297

Galβl-4GlcNAcβl-

3Galβl-4(Fucαl-

3)GlcNAcβl-3Galβl-

4(Fucαl-3)GlcNAcβ-

145 SpO -82 226 -244 -165 -1089 2249 81

Galβl-4GlcNAcβl-

3Galβl-4GlcNAcβl-

146 3Galβl-4GlcNAcβ-SpO 76 355 653 146 76 556 889

Galβl-4GlcNAcβl-

147 3Galβl-4GlcNAcβ-SpO 15 1854 55 242 -109 -177 376

Galβl-4GlcNAcβl-

148 3Galβl-4Glcβ-SpO -104 331 195 83 -243 933 1970

Galβl-4GlcNAcβl-

149 3Galβl-4Glcβ-Sp8 0 0 0 0 0 0 0

Galβl-4GlcNAcβl-

6(Galβl-3)GalNAcα-

150 Sp8 369 411 256 87 -234 48 1625

Galβl-4GlcNAcβl-

151 6GalNAcα-Sρ8 173 1371 715 818 -355 202 448

152 Galβl-4GlcNAcβ-SpO 985 831 4079 1679 453 928 3139

153 Galβl-4GlcNAcβ-Sp8 133 3159 1010 486 -140 1154 3072

154 Galβl-4Glcβ-SpO 388 1036 2889 1021 583 765 2238

155 Galβl-4Glcβ-Sp8 -80 202 1588 2259 -129 159 1043

GlcNAcαl-3Galβl-

156 4GlcNAcβ-Sp8 -204 950 244 548 -39 771 258

GlcNAcαl-6Galβl-

157 4GlcNAcβ-Sp8 -104 191 148 771 -876 1166 255

GlcNAcβl-2Galβl-

158 3GalNAcα-Sρ8 -296 -118 31 1158 -1267 963 -491

GlcNAcβl-

3(GlcNAcβl-

159 6)GalNAcα-Sp8 249 300 814 1265 -428 1134 415

GlcNAcβl-

3(GlcNAcβl-6)Galβl-

160 4GlcNAcβ-Sp8 -26 391 18 1167 167 841 825 GlcNAcβl-3GalNAcα-

161 Sp8 374 286 564 767 -365 -124 697

162 GlcNAcβl-3Galβ-Sp8 466 514 960 1985 714 -231 2743

GlcNAcβl-3Galβl-

163 3GalNAcα-Sρ8 140 843 828 1813 321 1889 1487

GlcNAcβl-3Galβl-

164 4GlcNAcβ-SpO 596 2210 1691 74 580 826 2875

GlcNAcβl-3Galβl-

165 4GlcNAcβ-Sp8 -118 228 416 1070 586 430 271

GlcNAcβl-3Galβl-

4GlcNAcβl-3Galβl-

166 4GlcNAcβ-SpO -74 296 -80 -216 -595 1 164

GlcNAcβl-3Galβl-

167 4Glcβ-SpO -93 2923 1477 1015 -71 738 2671

168 GlcNAcβl-4MDPLys 0 0 0 0 0 0 0

GlcNAcβl-

4(GlcNAcβl-

169 6)GalNAcα-Sp8 92 486 539 882 -259 1394 789

GlcNAcβl-4Galβl-

170 4GlcNAcβ-Sp8 0 0 0 0 0 0 0

171 (GlcNAcβl-4)6β-Sp8 -200 1970 478 769 -693 189 1401

172 (GlcNAcβl-4)5β-Sp8 -369 -107 -573 -339 -544 -580 461

GlcNAcβl-4GlcNAcβl-

173 4GlcNAcβ-Sp8 -370 4111 565 2141 404 1317 1580

GlcNAcβl-6(Galβl-

174 3)GalNAcα-Sp8 -74 -10 1124 1738 446 -193 913

GlcNAcβl-6GalNAcα-

175 Sp8 -107 -239 644 1357 60 1414 1134

GlcNAcβl-6Galβl-

176 4GlcNAcβ-Sp8 123 2493 754 741 127 976 641

177 Glcαl-4Glcβ-Sp8 -508 541 1185 4334 -83 878 2341

178 Glcαl-4Glcα-Sp8 521 383 251 3317 -513 1010 2231

Glcαl-6Glcαl-6Glcβ-

179 Sp8 -141 842 217 1545 -811 1201 1025

180 Glcβl-4Glcβ-Sp8 0 0 0 0 0 0 0

181 Glcβl-6Glcβ-Sp8 767 1037 1896 2572 -640 3551 1465

182 Sorbitol-Sp8 446 3988 1160 2271 48 539 3323

183 GlcAα-Sρ8 239 915 2578 1490 203 2282 6948

184 GlcAβ-Sp8 1061 1662 7208 5701 569 1802 11399

185 GlcAβl-3Galβ-Sp8 209 2419 2651 1274 -1016 -629 2596

186 GlcAβl-6Galβ-Sp8 658 1622 2179 2411 582 1586 4549

KDNα2-3Galβl-

187 3GlcNAcβ-SpO 338 2675 953 1268 -7 1176 3311

KDNα2-3Galβl-

188 4GlcNAcβ-SpO 1018 495 93 560 185 288 3984

Manαl-2Manαl-

189 2Manαl-3Manα-Sρ9 -13 2116 -413 219 -634 862 3555

Manαl-2Manαl-

3(Manαl-2Manαl-

190 6)Manα-Sρ9 379 35 1136 1372 -319 -723 2412

Manα 1 -2Manαl -3Manα-

191 Sp9 623 764 1358 671 856 321 5378

Manαl-6(Manαl-

2Manαl-3)Manαl-

6(Manα2Manαl-

3)Manβl-4GlcNAcβl-

192 4GlcNAcβ-N 804 403 1572 157 184 826 5909 Manαl-2Manαl- 6(Manαl-3)Manαl- 6(Manα2Manα2Manαl - 3)Manβl-4GlcNAcβl-

193 4GlcNAcβ-N 345 235 807 531 316 169 4519

Manαl-2Manαl- 2Manαl-3(Manαl- 2Manαl-3(Manαl- 2Manαl -6)Manαl - 6)Manβl-4GlcNAcβl-

-, ;494 4GlcNAcβ-N 415 680 833 -224 -149 -46 1228

Manαl-3(Manαl-

195 6)Manα-Sp9 0 0 0 0 0 0 0 Manαl-3(Manαl-

2Manαl-2Manαl-

196 6)Manα-Sp9 0 0 0 0 0 0 0 Manαl-6(Manαl-

3)Manαl-

6(Manα2Manαl-

3)Manβl-4GlcNAcβl-

197 4GlcNAcβ-N -17 296 484 -33 303 491 3920 Manαl-6(Manαl-

3)Manαl-6(Manαl- 3)Manβl-4GlcNAcβl-4

198 GlcNAcβ-N 398 3243 2224 521 300 111 5405

199 Man5_9mix N -445 -913 2852 1489 334 638 4257

200 Manβl-4GlcNAcβ-SρO 57 258 -678 -193 -1557 -1088 2398 Neu5Acα2-3(Galβl-

3GalNAcβl-4)Galβl-

201 4Glcβ-SpO 39 962 1573 1270 271 668 2789 Neu5Acα2-3Galβl-

202 3GalNAcα-Sp8 -99 1013 840 483 5 -113 702 NeuAcα2-8NeuAcα2-

8NeuAcα2-8NeuAcα2- 3(GalNAcβl-4)Galβl-

203. 4Glcβ-SpO 878 3229 659 1387 159 1560 6358

Neu5Acα2-8Neu5Acα2- 8Neu5Acα2- 3(GalNAcβl-4)Galβl-

204 4Glcβ-Sp0 80 2115 773 523 71 175 2051 Neu5Acα2-8Neu5Acα2-

8Neu5Acα2-3Galβl-

205 4Glcβ-SpO 160 1432 630 411 -112 285 1268 Neu5Acα2-8Neu5Acα2-

3(GalNAcβl-4)Galβl-

206 4Glcβ-SpO 226 953 708 682 -72 -64 1698 Neu5Acα2-8Neu5Acα2-

207 8Neu5Acα-Sp8 324 1456 1029 566 48 94 3687 Neu5Acα2-3(6-O-

Su)Galβl-4(Fucαl-

208 3)GlcNAcβ-Sp8 289 4481 2272 3182 -407 142 6159 Neu5Acα2-

3(GalNAcβl-4)Galβl- 1015

209 4GlcNAcβ-SpO -25 5 595 1001 -158 82 1248 Neu5Acα2-

3(GalNAcβl-4)Galβl-

210 4GlcNAcβ-Sp8 -2 347 526 145 -84 -167 1425 Neu5Acα2-

211 3(GalNAcβl-4)Galβl- 42 434 203 454 130 152 1740 4Glcβ-SpO

NeuAcα2-3(NeuAcα2-

3Galβl-3GalNAcβl-

212 4)Galβl-4Glcβ-SpO 382 894 214 212 -33 229 1656

Neu5Acα2-

3(Neu5Acα2-

213 6)GalNAcα-Sρ8 128 2140 2095 2209 232 601 6731

Neu5Acα2-3GalNAcα-

214 Sp8 -151 3368 413 467 -102 124 1170

Neu5Acα2-3GalNAcβl-

215 4GlcNAcβ-SρO -117 411 299 423 -405 -233 ^'-^'" 724

Neu5Acα2-3Galβl-

216 3(6OSO3)GlcNAc-Sp8 -45 2611 3303 2047 -657 -612 4267

Neu5Acα2-3Galβl-

3(Fucαl-4)GlcNAcβ-

217 Sp8 100 251 673 374 -4 648 635

NeuAcα2-3Galβl-

3(Fucαl-4)GlcNAcβl-

3Galβl-4(Fucαl-

218 3)GlcNAcβ SpO 0 0 0 0 0 0 0

Neu5Acα2-3Galβl-

3(Neu5Acα2-3Galβl-

219 4)GlcNAcβ-Sp8 132 954 172 289 -118 456 3360

Neu5Acα2-3Galβl-

220 3t6OSO3]GalNAcα-Sp8 140 3661 4231 3279 96 512 7674

Neu5Acα2-3Galβl-

3(Neu5Acα2-

221 6)GalNAcα-Sp8 467 1665 1982 2025 453 1279 5641

222 Neu5Acα2-3Galβ-Sp8 -273 1631 911 2127 261 587 3141

NeuAcα2-3Galβl-

3GalNAcβl-3Galαl-

223 4Galβl-4Glcβ-Sp0 138 442 547 911 -453 389 646

NeuAcα2-3Galβl-

3GlcNAcβl-3Galβl-

224 4GlcNAcβ-Sρ0 246 1436 425 106 -89 -60 502

Neu5Acα2-3Galβl-

225 3GlcNAcβ-SρO -26 1656 671 380 -264 233 975

Neu5Acα2-3Galβl-

226 3GlcNAcβ-Sp8 267 1155 282 537 107 . . 570 1470

Neu5Acα2-3Galβl-

227 4[6OSO3]GlcNAcβ-Sp8 549 4620 5345 6696 100 1012 13357

Neu5Acα2-3Galβl-

4(Fucαl-3)(6OSO3)

228 GlcNAcβ -Sp8 362 3646 2986 7990 251 1326 10743

Neu5Acα2-3Galβl-

4(Fucαl-3)GlcNAcβl-

3Galβl-4(Fucαl-

3)GlcNAcβl-3Galβl-

4(Fucαl-3) GlcNAcβ-

229 SpO -235 -95 2529 1837 -983 -209 2288

Neu5Acα2-3Galβl-

4(Fucαl-3)GlcNAcβ-

230 SpO -137 667 -171 -11 -261 260 1375

Neu5Acα2-3Galβl-

4(Fucαl-3)GlcNAcβ-

231 Sp8 32 697 429 729 -131 -158 852

Neu5Acα2-3Galβl-

4(Fucαl-3)GlcNAcβl-

232 3Galβ-Sp8 -8 367 -2342 -2493 -2624 -2156 -1027 Neu5Acα2-3Galβl-

4(Fucαl-3)GlcNAcβl-

233 3Galβl-4GlcNAcβ-Sp8 62 1342 335 616 181 447 3441

Neu5Acα2-3Galβl-

4GlcNAcβl-3Galβl-

234 4(Fucαl-3)GlcNAc-SpO 891 3929 913 707 -451 1568 2096

Neu5Acα2-3Galβl-

4GlcNAcβl-3Galβl-

235 4GlcNAcβ-SpO 281 1012 236 -104 --2222 9922 11886677

Neu5Acα2-3Galβl-

236 4GlcNAcβ-SpO 201 670 98 18 -118844 --116600 552211

Neu5Acα2-3Galβl-

237 4GlcNAcβ-Sp8 128 445 799 66 -331100 --118800 554400

Neu5Acα2-3Galβl-

4GlcNAcβl-3Galβl-

238 4GlcNAcβ-SpO -82 1244 2017 2152 -221111 446633 224477

Neu5Acα2-3Galβl-

239 4Glcβ-Sp0 202 2147 2772 2459 2288 881177 55330022

Neu5Acα2-3Galβl-

240 4Glcβ-Sp8 0 0 0 0 00 00 00

Neu5Acα2-6(Galβl-

241 3)GalNAcα-Sp8 406 1307 280 650 -118899 1155 889988

Neu5Acα2-6GalNAcα-

242 Sp8 1012 3180 3390 1843 --4444 --77 22441188

Neu5Acα2-6GalNAcβl-

243 4GlcNAcβ-SpO -147 1063 1012 166 --44 --8811 773366

Neu5Acα2-6Galβl- 1054 1594

244 4[6OSO3]GlcNAcβ-Sp8 2526 7600 8 7 662222 11664411 1199662277

Neu5Acα2-6Galβl-

245 4GlcNAcβ-SpO 257 2413 1981 1371 -227722 331100 11116600

Neu5Acα2-6Galβl-

246 4GlcNAcβ-Sp8 117 805 1202 1109 88 111177 m 112i7

Neu5Acα2-6Galβl-

4GlcNAcβl-3Galβl-

4(Fucαl-3)GlcNAcβl-

3Galβl-4(Fucαl-

247 3)GlcNAcβ-Sp0 9 1694 1376 823 122 981 2427

Neu5Acα2-6Galβl-

4GlcNAcβl-3Galβl-

248 4GlcNAcβ-SpO 346 311 307 490 15 195 1313

Neu5Acα2-6Galβl-

249 4Glcβ-SpO 121 1957 -3523 -2000 -3041 -2524 2581

Neu5Acα2-6Galβl-

250 4Glcβ-Sp8 1081 846 2738 599 726 893 6497

251 Neu5Acα2-6Galβ-Sp8 516 747 2044 1185 366 1208 5781

Neu5Acα2-8Neu5Acα-

252 Sp8 699 2119 2226 2703 -261 1175 . 5482

Neu5Acα2-8Neu5Acα2-

253 3Galβl-4Glcβ-Sp0 223 1843 2440 2038 -10 470 1708

Neu5Acβ2-6GalNAcα-

254 Sp8 -20 3412 823 1248 -419 -102 599

Neu5Acβ2-6Galβl-

255 4GlcNAcβ-Sp8 324 1565 958 945 84 516 1555

Neu5Acβ2-6(Galβl-

256 3)GalNAcα-Sp8 741 674 -556 -834 -1024 267 2506

Neu5Gcα2-3Galβl-

3(Fucαl-4)GlcNAcβ-

257 SpO 349 1032 772 290 -341 -27 387 Neu5Gcα2-3Galβl-

258 3GlcNAcβ-SpO 330 2004 3122 1746 437 1120 5152

Neu5Gcα2-3Galβl-

4(Fucαl-3)GlcNAcβ-

259 SpO -96 1601 558 -166 -56 -73 2396

Neu5Gcα2-3Galβl-

260 4GlcNAcβ-SpO 612 2740 1649 1254 278 744 5341

Neu5Gcα2-3Galβl-

261 4Glcβ-SpO 321 526 -4 -4 -23 -218 3559

Neu5Gcα2-6GalNAcα-

262 SpO -130 1419 1375 1223 -149 -183 4584

Neu5Gcα2-6Galβl-

263 4GlcNAcβ-SpO 255 1457 2543 1168 1101 891 5708

264 Neu5Gcα-Sρ8 807 1171 2787 351 -48 529 2508

[3OSO3]Galβl-4(Fucαl-

265 3)(6OSO3)Glc-Sp0 287 2973 3432 2967 -210 517 6216

[3OSO3]Galβl-4(Fucαl-

266 3)Glc-Sp0 1641 1235 1867 -407 108 2202 9983

[3OSO3]Galβl-4[Fucαl- 1065 1222

267 3][6OSO3]GlcNAc-Sp8 2525 8386 6 5 1151 5481 22620

[3OSO3]Galβl-4[Fucαl-

268 3]GlcNAc-Sp0 -28 1983 1783 394 91 -264 3404

Fucαl-2[6OSO3]Galβl-

269 4GIcNAc-SpO 491 2316 2175 727 467 1178 7060

Fucαl-2Galβl-

270 4[6OSO3]GlcNAc-Sp8 744 1308 2069 929 -9 405 8029

Fucαl-2[6OSO3]Galβl- 1328

271 4[6OSO3]Glc-Sp0 3158 9728 8466 1 3666 7237 29583

Fucαl-2-(6OSO3)-

272 Galβl-4Glc-SpO 347 2483 2890 298 255 306 6644

Fucαl-2-Galβl-

273 4[6OSO3]Glc-Sp0 660 885 4507 1504 575 2162 12180

Galβl-3(Fucαl-

4)GlcNAcβl-3Galβl-

3(Fucαl-4)GlcNAcβ-

274 SpO -123 261 369 766 -339 29 923

Galβl-3-(Galβl-

275 4GlcNacβl-6)GalNAc-T 20 1171 355 156 -297 -322 906

Galβl-3(GlcNacβl-

276 6)GaINAc-T 634 954 1962 1190 -559 -138 1223

Galβl-3-(Neu5Aα2-

3Galβl-4GlcNacβl-

277 6)GalNAc-T -364 5191 1743 1457 -958 -882 1010

278 Galβl-3GalNAc-T 511 799 2026 2032 211 218 10079

Galβl-3GlcNAcβl-

279 3Galβl-3GlcNAcβ-Sp0 173 271 387 487 6 -74 270

Galβl-4[Fucαl-

280 3][6OSO3]GlcNAc-Sp0 396 1332 609 1062 -21 679 4531

Galβl-4[Fucαl-

281 3][6OSO3]Glc-Sp0 554 1516 1484 400 -439 457 2771

Galβl-4(Fucαl-

3)GlcNAcβl-3Galβl-

3(Fucαl-4)GlcNAcβ-

282 SpO 536 650 2066 666 -336 221 5489

Galβl-4GlcNAcβl-

283 3Galβl-3GlcNAcβ-Sp0 -73 337 172 37 -286 -258 549

Neu5Acα2-3Galβl-

3GlcNAcβl-3Galβl-

284 3GlcNAcβ-SpO -169 1350 1001 2487 -348 681 1113 Neu5Acα2-3Galβl- 4GlcNAcβl-3Galβl- 285 3GlcNAcβ-SpO -90 -18 1376 -1266 -1495 -1490 -683

Table 26. Determination of specific glycan binding by 6RG::His, 4WC::His, 6RG::4WC::His and 4WC::6RG::His. Relative Fluorescence Units (RFU) detected at each glycan has been adjusted for specific and non-specific antibody background binding.

Analysis of 4WC and 6RG glycan binding profiles

The insect cell expressed recombinant apyrases 4WC and 6RG preferentially bind sulfated and phosphorylated carbohydrates at pH5.0

Both 4WC and 6RG demonstrated a strong binding preference to specific sulfated galactose glycans located between glycans 26 and 45. At low pH (5.0), 4WC demonstrated a modest preference to glycan 26 ([3OSO3][6OSO3]Galβ1-4[6OSO3]GlcNAcβ-Sp0), while 6RG bound almost equally to glycans 26, 27 ([3OSO3][6OSO3]Galβ1-4GlcNAcβ-Sp0) and 39 ([4OSO3][6OSO3]Galβ1 -4GlcNAcβ- SpO).

Binding to singly sulfated reducing sugars ([3OSO3]Gal, [4OSO3]Gal or [6OSO3]Gal) that were not linked to a second sulfated sugar was comparatively poor as seen by the somewhat reduced binding to the sulfated glycans 244 (Neu5Acα2-6Galβ1-4[6OSO3]GlcNAcβ-Sp8), 267 ([3OSO3]Galβ1-4[Fucα1- 3][6OSO3]GlcNAc-Sp8) and 271 (Fucα1-2[6OSO3]Galβ1-4[6OSO3]Glc-Sp0. A further reduction in binding affinity was measured for the phosphorylated glycan 41 (6-H2PO3Manα-Sp8) and further reductions were determined for glycans 227 (Neu5Acα2-3Galβ1-4[6OSO3]GlcNAcβ-Sp8) and 228 (Neu5Acα2-3Galβ1-4(Fucα1-3)(6OSO3) GlcNAcβ -Sp8).

6RG bound relatively weakly to only one non-sulfated glycan to any degree, this was glycan 209 (Neu5Acα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ-SpO) and this was only bound at pH 5. In comparison 4WC bound relatively weakly to 2 different non sulfated glycans (177, Glcα1-4Glcβ-Sp8; 184, GlcAβ- Sp8), where binding to 177 only occurred at pH 5 whereas binding to 184 was slightly elevated at pH 7.2. Overall, both clones demonstrated strong binding to the sulfated glycans 26, 27, 29, 30, 35, 39 and 45 with the highest affinity occurring when the reducing galactose was disulfated (at either the 3rd and 6th carbon, or the 4th and 6th carbohydrate); the next highest affinity binding was to singly sulfated reducing galactose (at either the 3rd or 6th carbon) but only when this was linked (β1-4) to a second sulfated carbohydrate consisting of either [60S03]N-acetylglucosamine or [6OSO3]glucose. Generally, at pH 5.0 the binding of 4WC and 6RG to the specific glycans above was higher (depending on the glycan) compared to binding at pH 7.2. The increase for 4WC was between 20- 50% whereas for 6RG it was between 200-300%.

Analysis of chimeric 4WC::6RG and 6RG::4WC glycan binding

The N-terminal half of the protein appears to be predominantly responsible for determining glycan specificity as well as response to pH

On the whole the 4WC::6RG clone closely followed 4WC while 6RG::4WC followed 6RG both in terms of the pattern of sulfated glycan binding and the response to pH. The data suggests that for apyrase binding to the sulfated carbohydrates the N-terminal half of the protein is predominantly responsible for determining glycan specificity as well as carbohydrate binding response to pH.

Generation of chimeras changes glycan binding specificity

While the binding of the chimeras and their parents to the sulfated carbohydrates was a predominant feature, the generation of the chimeras altered the binding profile to a number of key glycans, as seen by:

^• the elevated affinity (compared to both parents) of 4WC::6RG to the glycans 37, 38, 40, 42, 43 and 44 which all contain a single sulfate group in the form of [6OSO3]Galβ1-4 at the reducing end and the glycans 227 (Neu5Acα2-3Galβ1-4[6OSO3]GlcNAcβ-Spδ), 228 (Neu5Acα2-3Galβ1-4(Fucα1- 3)(6OSO3) GlcNAcβ -Sp8) and 244 (Neu5Acα2-6Galβ1-4[6OSO3]GlcNAcβ-Sp8) which all contain a single sulfate group in the form of [6OSO3]GlcNAc. • the elevated affinity (compared to both parents) of 4WC::6RG to the predominantly sialic acid glycans 203 (NeuAcα2-8NeuAcα2-8NeuAcα2-8NeuAcα2-3(GalNAcβ1-4)Galβ1-4Glcβ-Sp0), 207 (Neu5Acα2-8Neu5Acα2-8Neu5Acα-Sp8), 208 (Neu5Acα2-3(6-O-Su)Galβ1-4(Fucα1-3)GlcNAcβ-Sp8), 213 (Neu5Acα2-3(Neu5Acα2-6)GalNAcα-Sp8) and 220 (Neu5Acα2-3Galβ1-3[6OSO3]GalNAcα-Sp8). The binding affinity for each of these glycans appeared to be higher at pH 5.0 when compared with pH 7.2.

the reduced binding of 6RG::4WC to glycan 244 (Neu5Acα2-6Galβ1-4[6OSO3]GlcNAcβ-Sp8) compared to the 6RG parent clone.

This indicates that the there is some influence of the C-terminal half on the carbohydrate binding characteristics. These results are virtually the reverse of the location of apyrase substrate specificity determination where we found this was primarily determined by the C-terminus with small modifications made by the N-terminus.

Comparison with 7WC glycan binding

7WC::His produced in the prokaryotic expression system was also analysed for specific glycan binding by Core-H. The slides were incubated in either: 5OmM Acetate buffer pH 5.0, 3mM calcium chloride,

0.05% Tween 20, 1% BSA, 5mM ATP; or 5OmM Tris-HCI pH 8.0, 3mM calcium chloride, 0.05% Tween

20, 1% BSA, 5mM ATP. Following several washes in the appropriate buffer the were probed with rabbit anti-7WC @ 1/500. Antibody binding was detected using FITC-anti-rabbit IgG (supplied by

Core-H). Carbohydrate binding was then determined indirectly by Relative Fluorescence Units (RFU) emitted by the FITC labelled secondary antibody. The 7WC glycan binding data corrected for specific antibody binding and non specific antibody binding is shown in Table 27.

RTU RFU

{Jf^an Glycan Name 7WC 7WC

(pH5) (pH8)

1 Alphal-acid glycoprotein (AGP) 33580 1581

2 AGP-A (AGP ConA flowthrough) 13615 2107

3 AGP-B (AGP ConA bound) 0 0

4 Ceruloplasmiα 27559 12700 Fibrinogen 3457 136

Transferrin 0 0 α-D-Gal-Sρ8 775 1094 α-D-Glc-Sp8 2396 3710 α-D-Man-Sρ8 1830 617 α-GalNAc-Sρ8 592 982 α-L-Fuc-Sp8 883 964 α-L-Fuc-Sρ9 383 300 α-L-Rhα-Sp8 580 . 953 α-Neu5Ac-Sp8 1101 361

Neu5Acαl-2-Sρ82 610 127 b-Neu5Ac-Sρ8 842 409 β-D-Gal-Sp8 669 552 β-D-Glc-Sp8 2273 2624 β-D-Man-Sρ8 239 -206 β-GalNAc-Sp8 1117 1108 β-GlcNAc-SpO 1000 1592 β-GlcNAc-Sp8 307 965 b-GlcN(Gc)-Sp8 -140 -15

(Galβl-4GlcNAcβ)₂-3,6-GalNAcα-Sp8 -1172 -563

(GlcNAcbl-3(GlcNAcbl-6)GlcNAcbl-4)GlcNAc-

3236 10126 Sp8

[3OSO3][6OSO3]Galbl-4[6OSO3]GlcNAcb-Sp0 25116 259

[3OSO3][6OSO3]Galbl-4GlcNAcb-Sp0 4172 216

[3OSO3]Galbl-4Glcb-Sp8 1050 445

[3OSO3]Galβl-4(6OSO3)Glcβ-Sp0 2370 1227

[3OSO3]Galβl-4(6OSO3)Glcβ-Sp8 2418 -90

[3OSO3]Galβl-3(Fucαl-4)GlcNAcβ-Sp8 269 665

[3OSO3]Galβl-3GalNAcα-Sp8 1644 2562

[3OSO3]Galβl-3GlcNAcβ-Sp8 417 1028

[3OSO3]Galβl-4(Fucαl-3)GlcNAcβ-Sp8 212 302

[3OSO3]Galbl-4[6OSO3]GlcNAcb-Sp8 4028 477

[3OSO3]Galβl-4GlcNAcβ-Sp0 627 409

[3OSO3]Galbl-4GlcNAcb-Sp8 841 285

[3OSO3]Galβ-Sp8 562 -5

[4OSO3][6OSO3]Galbl-4GlcNAcb-Sp0 4001 1168

[4OSO3]Galbl-4GlcNAcb-Sp8 264 426

6-H₂PO₃Manα-Sρ8 1584 1056

[6OSO3]Galβl-4Glcβ-Sp0 677 1205

[6OSO3]Galβl-4Glcβ-Sp8 4144 1519

[6OSO3]Galβl-4GlcNAcβ-Sp8 1249 1701

[6OSO3]Galbl-4[6OSO3]Glcb-Sp8 3850 2757

NeuAcα2-3[6OSO3]Galβl-4GlcNAcβ-Sρ8 468 19

[6OSO3]GlcNAcβ-Sp8 799 1540

9NAcNeu5Aca-Sp8 2970 191

9NAcNeu5Aca2-6Galbl-4GlcNAcb-Sp8 158 186

Manal-3(Manal-6)Manbl-4GlcNAcbl-4GlcNAcb-

-940 -470 GIy - ₁ GlcNAcbl-2Manal-3(GlcNAcbl-2Manal-

680 3850 6)Manbl-4GlcNAcbl-4GlcNAcb-Gly Galbl-4GlcNAcbl-2Manal-3(Galbl-4GlcNAcbl-

1446 2Manal-6)Manbl-4GlcNAcbl-4GlcNAcb-Gly 73 Neu5Aca2-3Galbl-4GlcNAcbl-2Manal-

53 3(Neu5Aca2-3Galbl-4GlcNAcbl-2Manal- 43 59 6)Manbl-4GlcNAcbl-4GlcNAcb-Gly Neu5Aca2-3Galbl-4GlcNAcbl-2Manal-

54 3(Neu5Aca2-3Galbl-4GlcNAcbl-2Manal- 99 267 6)Manbl-4GlcNAcbl-4GlcNAcb-Sρ8

55 Fucal-2Galbl-3GalNAcbl-3Gala-Sρ9 180 247

„ Fucal^Galbl-SGalNAcbl-SGalal^Galbl^Glcb-

1390 103

⁵⁶ Sp9

57 Fucαl-2Galβl-3(Fucαl-4)GlcNAcβ-Sp8 406 579

58 Fucαl-2Galβl-3GalNAcα-Sp8 217 705

_₀ Fucal-2Galbl-3GalNAcbl-4(Neu5Aca2-3)Galbl-

413 249

4GlCb-SpO ,„ Fucαl-2Galβl-3GalNAcβl-4(Neu5Acα2-3)Galβl- 106 217

4Glcβ-Sp9

61 Fucαl-2Galβl-3GlcNAcβl-3Galβl-4Glcβ-SplO 450 615

62 Fucαl-2Galβl-3GlcNAcβl-3Galβl-4Glcβ-Sp8 711 977

63 Fucαl-2Galβl-3GlcNAcβ-SpO 304 539

64 Fucαl-2Galβl-3GlcNAcβ-Sp8 225 89

,, Fucal-2Galbl-4(Fucal-3)GlcNAcbl-3Galbl- _{1 f?} . . _u

4(Fucal-3)GlcNAcb-SpO Fucal-2Galbl-4(Fucal-3)GlcNAcbl-3Galbl-

66 4(Fucal-3)GlcNAcbl-3Galbl-4(Fucal- -222 321 3)GlcNAcb-SpO

67 Fucαl-2Galβl-4(Fucαl-3)GlcNAcβ-SpO -241 -327

68 Fucαl-2Galβl-4(Fucαl-3)GlcNAcβ-Sp8 166 -52

69 Fucαl-2Galβl-4GlcNAcβl-3Galβl-4GlcNAc-SρO 26 74 Fucal-2Galbl-4GlcNAcbl-3Galbl-4GlcNAcbl- -558 -637

' 3Galbl-4GlcNAcb-SpO

71 Fucαl-2Galβl-4GlcNAcβ-SpO -31 -23

72 Fucαl-2Galβl-4GlcNAcβ-Sp8 -110 336

73 Fucαl-2Galβl-4Glcβ-SpO 2820 554

74 Fucαl-2Galβ-Sρ8 1015 -448

75 Fucal-2GlcNAcb-Sp8 46 323

76 Fucαl-3GlcNAcβ-Sp8 -108 767

77 Fucαl-4GlcNAcβ-Sp8 -30 -296

78 Fucbl-3GlcNAcb-Sp8 525 846

79 GalNAcal-3(Fucal-2)Galbl-3GlcNAcb-SpO -527 -91

_0Λ GalNAcal-3(Fucal-2)Galbl-4(Fucal-3)GlcNAcb-

48 8

⁸⁰ SpO

81 GalNAcal-3(Fucal-2)Galbl-4GlcNAcb-SpO -604 -505

82 GalNAcαl-3(Fucαl-2)Galβl-4GlcNAcβ-Sp8 -384 -253

83 GalNAcal-3(Fucal-2)Galbl-4Glcb-SρO -340 219

84 GalNAcαl-3(Fucαl-2)Galβ-Sp8 351 183

85 GalNAcαl-3GalNAcb-Sp8 1061 541

86 GalNAcαl-3Galb-Sp8 219 1283

87 GalNAcal-4(Fucal-2)Galbl-4GlcNAcb-Sp8 620 871

88 GalNAcbl-3GalNAca-Sp8 915 2891

89 GalNAcbl-3(Fucal-2)Galb-Sp8 967 1001

90 GalNAcbl-3Galal-4Galbl-4GlcNAcb-SpO 338 537 91 GalNAcbl-4(Fucal-3)GlcNAcb-SpO 2090 1251

92 GalNAcβl-4GlcNAcβ-SpO 61 970

93 GalNAcβl-4GlcNAcβ-Sρ8 835 1236

94 Galαl-2Galβ-Sp8 -63 -3

95 Galal-3(Fucal-2)Galbl-3GlcNAcb-SpO 531 527

96 Galal-3(Fucal-2)Galbl-4(Fucal-3)GlcNAcb-SpO -25 280

97 Galal-3(Fucal-2)Galbl-4GlcNAc-SpO 405 939

98 Galal-3(Fucal-2)Galbl-4Glcb-SpO 772 627

99 Galαl-3(Fucαl-2)Galβ-Sρ8 0 0

100 Galal-3(Galal-4)Galbl-4GlcNAcb-Sp8 761 762

101 Galal-3GalNAca-Sp8 709 1522

102 Galαl-3GalNAcβ-Sp8 535 988

103 Galαl-3Galβl-4(Fucαl-3)GlcNAcβ-Sp8 41 605

104 Galal-3Galbl-3GlcNAcb-SpO 640 208

105 Galαl-3Galβl-4GlcNAcβ-Sp8 1196 117

106 Galαl-3Galβl-4Glcβ-SpO 4146 737

107 Galαl-3Galβ-Sρ8 -97 468

108 Galal-4(Fucal-2)Galbl-4GlcNAcb-Sp8 395 289

109 Galαl-4Galβl-4GlcNAcβ-SpO -90 -144

110 Galαl-4Galβl-4GlcNAcβ-Sp8 159 349

111 Galαl-4Galβl-4Glcβ-SpO -300 598

112 Galαl-4GlcNAcb-Sp8 866 1405

113 Galal-6Glcb-Sp8 0 0

114 Galβl-2Galβ-Sϋ8 -511 628

„ „ Galbl-3(Fucal-4)GlcNAcbl-3Galbl-4(Fucal- ₀ . . .. ..

^U5 3)GlcNAcb-Sp0 ^{2 /45 / 141}

₁₁₆ Galbl-3(Fucal-4)GlcNAcbl-3Galbl-4GlcNAcb- _{1?23 y33} SpO

117 Galβl-3(Fucαl-4)GlcNAc-SpO 1345 1042

118 Galβl-3(Fucαl-4)GlcNAc-Sp8 1057 1604

119 Galβl-3(Fucαl-4)GlcNAcβ-Sp8 -140 -201

120 Galβl-3(Galβl-4GlcNAcβl-6)GalNAcα-Sp8 4721 1999

121 Galβl-3(GlcNAcβl-6)GalNAcα-Sp8 1439 5097

122 Galbl-3(Neu5Aca2-6)GalNAca-Sp8 764 1321

123 Galbl-3(Neu5Acb2-6)GalNAca-Sp8 896 1484

. , . Galbl-3(Neu5Aca2-6)GlcNAcbl-4Galbl-4Glcb-

3480 1615

^1/4 SpIO

125 Galβl-3GalNAcα-Sp8 \iπ 3369

126 Galβl-3GalNAcβ-Sp8 IAYl 4713

127 Galbl-3GalNAcbl-3Galal-4Galbl-4Glcb-SpO 373 990

, _₀ Galbl-3GalNAcbl-4(Neu5Aca2-3)Galbl-4Glcb-

512 1011 SpO

129 Galβl-3GalNAcβl-4Galβl-4Glcβ-Sp8 2093 2808

130 Galβl-3Galβ-Sp8 1606 4912

131 Galbl-3GlcNAcbl-3Galbl-4GlcNAcb-SpO 1683 532

132 Galβl-3GlcNAcβl-3Galβl-4Glcβ-SplO 799 1111

133 Galβl-3GlcNAcβ-SpO 519 935

134 Galβl-3GlcNAcβ-Sp8 176 1185

135 Galβl-4(Fucαl-3)GlcNAcb-SpO 744 972

136 Galβl-4(Fucαl-3)GlcNAcb-Sp8 256 301 _{i r7} Galbl-4(Fucal-3)GlcNAcbl-4Galbl-4(Fucal- _ .,

^{1J /} 3)GlcNAcb-SpO ^{iyi4 /435} , Galβl-4(Fucαl-3)GlcNAcβl-4Galβl-4(Fucαl-

⁸ 3)GlcNAcβl-4Galβl-4(Fucαl-3)GlcNAcβ-SpO ^y/V

139 Galβl-4[6OSO3]Glcβ-Sp0 3588 6090

140 Galβl-4[6OSO3]Glcβ-Sp8 1838 2311

_AΛ Galbl-4GalNAcal-3(Fucal-2)Galbl-4GlcNAcb- 860 1516

¹⁴¹ Sp8

, ₄₀ Galbl-4GalNAcbl-3(Fucal-2)Galbl-4GlcNAcb- 165 1095

¹⁴² Sp8

_{Λ 4}, Galbl-4GlcNAcbl-3(Galbl-4GlcNAcbl- 591 550

6)GalNAca-Sp8

144 Galβl-4GlcNAcβl-3GalNAcα-Sp8 255 212

Galbl-4GlcNAcbl-3Galbl-4(Fucal-3)GlcNAcbl- 160 472

3Galbl-4(Fucal-3)GlcNAcb-SpO

_14Λ Galβl-4GlcNAcβl-3Galβl-4GlcNAcβl-3Galβl- „ , .

¹⁴⁰ 4GlcNAcβ-SpO

147 Galβl-4GlcNAcβl-3Galβl-4GlcNAcβ-SpO -2151 -2289

148 Galβl-4GlcNAcβl-3Galβl-4Glcβ-SpO 639 1293

149 Galβl-4GlcNAcβl-3Galβl-4Glcβ-Sp8 520 958

150 Galβl-4GlcNAcβl-6(Galβl-3)GalNAcα-Sρ8 1897 2058

151 Galβl-4GlcNAcβl-6GalNAcα-Sp8 1253 1804

152 Galβl-4GlcNAcβ-SpO -51 18

153 Galβl-4GlcNAcβ-Sp8 716 1146

154 Galβl-4Glcβ-SpO 1342 2632

155 Galβl-4Glcβ-Sρ8 2784 3705

156 GlcNAcal-3Galbl-4GlcNAcb-Sp8 899 1960

157 GlcNAcal-6Galbl-4GlcNAcb-Sp8 2030 3344

158 GlcNAcβl-2Galβl-3GalNAcα-Sp8 373 1030

159 GlcNAcβl-3(GlcNAcβl-6)GalNAcα-Sp8 1346 4636

160 GlcNAcβl-3(GlcNAcβl-6)Galbl-4GlcNAcb-Sp8 1564 4072

161 GlcNAcβl-3GalNAcα-Sρ8 832 594

162 GlcNAcbl-3Galb-Sp8 1070 2848

163 GlcNAcbl-3Galbl-3GalNAca-Sp8 2069 3379

164 GlcNAcβl-3Galβl-4GlcNAcβ-SpO 87 439

165 GlcNAcbl-3Galbl-4GlcNAcb-Sp8 576 930

_Λ , , GlcNAcbl-SGalbMGlcNAcbl-SGalbl- -45 674

¹⁰⁰ 4GlcNAcb-SpO

167 GlcNAcβl-3Galβl-4Glcβ-SpO 2258 3535

168 GlcNAcb 1 -4MDPLys (bacterial cell wall) 0 0

169 GlcNAcbl-4(GlcNAcbl-6)GalNAca-Sρ8 2132 4403

170 GlcNAcbl-4Galbl-4GlcNAcb-Sp8 511 1200

171 (GlcNAcbl-4)₆β-Sp8 840 1478

172 (GlcNAcbl-4)₅β-Sp8 -95 644

173 GlcNAcβl-4GlcNAcβl-4GlcNAcβ-Sp8 1052 2155

174 GlcNAcβl-6(Galβl-3)GalNAcα-Sp8 2962 3780

175 GlcNAcβl-6GalNAcα-Sp8 2511 3451

176 GlcNAcbl-6Galbl-4GlcNAcb-Sp8 1097 2496

177 Glcαl-4Glcβ-Sp8 4987 9304

178 Glcαl-4Glca-Sp8 4766 12420 179 Glcαl-6Glcαl-6Glcβ-Sp8 6875 7675

180 Glcbl-4Glcb-Sp8 7035 10510

181 Glcbl-6Glcb-Sp8 6965 10960

182 G-ol-amine 1834 225

183 GlcAa-Sp8 1393 2245

184 GlcAb-Sp8 720 2369

185 GlcAbl-3Galb-Sp8 0 0

186 GlcAbl-6Galb-Sp8 1900 -294

187 KDNα2-3Galβl-3GlcNAcβ-SpO 2116 2198

188 KDNα2-3Galβl-4GlcNAcβ-SpO 584 582

189 Manal-2Manal-2Manal-3Mana-Sp9 620 1703

190 Manal-2Manal-3(Manal-2Manal-6)Mana-Sp9 2416 8070

191 Manal-2Manal-3Mana-Sp9 -60 313 Manal-6(Manal-2Manal-3)Manal-

192 6(Manα2Manαl-3)Manbl-4GlcNAcbl-4GlcNAcb- 1285 2815 Asn

Manαl-2Manαl-6(Manαl-3)Manαl-

193 6(Manα2Manα2Manαl-3)Manβl-4GlcNAcβl- 182 5002 4GlcNAcβ-Asn Manαl-2Manαl-2Manαl-3(Manαl-2Manαl-

194 3(Manαl-2Manαl-6)Manαl-6)Manβl- 1296 3272 4GlcNAcβl-4GlcNAcβ-Asn

195 Manαl-3(Manαl-6)Manα-Sp9 112 341

196 Manal-3(Manal-2Manal-2Manal-6)Mana-Sρ9 0 0 . g₇ Manαl-6(Manαl-3)Manαl-6(Manα2Manαl-

3)Manβl-4GlcNAcβl-4GlcNAcβ-Asn -688 269

₁ g_o Manal-6(Manal-3)Manal-6(Manal-3)Manbl- 602 509 4GlcNAcbl-4 GlcNAcb-Asn

199 Man5-9mix-Asn 941 1952

200 Manbl-4GlcNAcb-SpO 845 1638

_9m Neu5Aca2-3(Galbl-3GalNAcbl-4)Galbl-4Glcb-

-340 204

SpO

202 Neu5Aca2-3Galbl-3GalNAca-Sp8 151 476 -„, NeuAca2-8NeuAca2-8NeuAca2-8NeuAca2- 269 396

3(GalNAcbl-4)Galbl-4Glcb-SpO

Neu5Aca2-8Neu5Aca2-8Neu5Aca2-3(GalNAcbl-

851 460 υ 4)Galbl-4Glcb-SpO

Neu5Aca2-8Neu5Aca2-8Neu5Acα2-3Galβl- 452 821 υ^:> 4Glcβ-SpO

»_n, Neu5Aca2-8Neu5Acα2-3(GalNAcβl-4)Galβl- 230 413 υ⁰ 4Glcβ-SpO

207 Neu5Acα2-8Neu5Acα2-8Neu5Acα-Sp8 2225 -2080

_OΛQ Neu5Acα2-3(6-O-Su)Galβl-4(Fucαl-3)GlcNAcβ-

626 78

²⁰⁸ Sp8

209 Neu5Aca2-3(GalNAcbl-4)Galbl-4GlcNAcb-SpO 5815 847

210 Neu5Aca2-3(GalNAcbl-4)Galbl-4GlcNAcb-Sp8 1015 229

211 Neu5Acα2-3(GalNAcβl-4)Galβl-4Glcβ-SpO 174 75

₀₁ _ NeuAca2-3(NeuAca2-3Galbl-3GalNAcbl- _{1 δ} , . . .

²¹² 4)Galbl-4Glcb-SpO ^{185 144}

213 Neu5Acα2-3(Neu5Acα2-6)GalNAcα-Sp8 -25 -58

214 Neu5Acα2-3GalNAcα-Sρ8 845 1265

215 Neu5Aca2-3GalNAcbl-4GlcNAcb-SpO 1743 -1170

216 Neu5Acα2-3Galβl-3(6OSO3)GlcNAc-Sp8 719 450 217 Neu5Acα2-3Galbl-3(Fucαl-4)GlcNAcβ-Sp8 2015 299

_{91 S} NeuAca2-3Galbl-3(Fucal-4)GlcNAcbl-3Galbl- „

4(Fucal-3)GlcNAcb-SpO ^υ

_{01 Q} Neu5Acα2-3Galβl-3(Neu5Acα2-3Galβl- _{1 /l 0 oo}

²¹⁹ 4)GlcNAcβ-S_P8 ^{"142 "88}

220 Neu5Aca2-3Galbl-3[6OSO3]GalNAca-Sp8 396 138

221 Neu5Acα2-3Galβl-3(Neu5Acα2-6)GalNAcα-Sp8 1223 3

222 Neu5Aca2-3Galb-Sp8 -1591 -1844

225 Neu5Acα2-3Galβl-3GlcNAcβ-SρO 497 604

226 Neu5Acα2-3Galβl-3GlcNAcβ-Sρ8 2008 -1726

227 Neu5Aca2-3Galbl-4[6OSO3]GlcNAcb-Sp8 134 86 _ooe Neu5Acα2-3Galβl-4(Fucαl-3)(6OSO3)GlcNAcβ- . , , , n⁶- Sp8 ^{4/ 'i ib}

Neu5Acα2-3Galβl-4(Fucαl-3)GlcNAcβl-3Galβl-

229 4(Fucαl-3)GlcNAcβl-3Galβl-4(Fucαl- 0 0 3)GlcNAcβ-SpO

230 Neu5Acα2-3Galβl-4(Fucαl-3)GlcNAcβ-SpO 129 147

231 Neu5Acα2-3Galβl-4(Fucαl-3)GlcNAcβ-Sp8 -205 -195 ,„ Neu5Aca2-3Galbl-4(Fucal-3)GlcNAcbl-3Galb-

Sp8 -79 -201 ,„ Neu5Aca2-3Galbl-4(Fucal-3)GlcNAcbl-3Galbl- -534 -640

4GlcNAcb-Sp8 0_% . Neu5Aca2-3Galbl-4GlcNAcbl-3Galbl-4(Fucal- 909 1087

3)GlcNAc-SpO -, , Neu5Acα2-3Galβl-4GlcNAcβl-3Galβl- 70 52

4GlcNAcβl-3Galβl-4GlcNAcβ-SpO

236 Neu5Acα2-3Galβl-4GlcNAcβ-SρO 164 -80

237 Neu5Acα2-3Galβl-4GlcNAcβ-Sp8 -792 -728 ₉__s Neu5Aca2-3Galbl-4GlcNAcbl-3Galbl- 2014

4GlcNAcb-SpO 1149

239 Neu5Acα2-3Galβl-4Glcβ-SpO 2061 654

240 Neu5Acα2-3Galβl-4Glcβ-Sp8 8254 5750

241 Neu5Acα2-6(Galβl-3)GalNAcα-Sp8 490 380

242 Neu5Acα2-6GalNAcα-Sp8 688 133

243 Neu5Aca2-6GalNAcbl-4GlcNAcb-SpO 1587 1118

244 Neu5Aca2-6Galbl-4[6OSO3]GlcNAcb-Sp8 -944 -1527

245 Neu5Acα2-6Galβl-4GlcNAcβ-SρO 1704 1910

246 Neu5Acα2-6Galβl-4GlcNAcβ-Sp8 1863 -138

₉ . Neu5Aca2-6Galbl-4GlcNAcbl-3Galbl-4(Fucal- 419 780

3)GlcNAcbl-3Galbl-4(Fucal-3)GlcNAcb-SpO . Neu5Aca2-6Galbl-4GlcNAcbl-3Galbl- 180 215 4GlcNAcb-SpO

249 Neu5Acα2-6Galβl-4Glcβ-SpO 373 417

250 Neu5Acα2-6Galβl-4Glcβ-Sp8 1561 370

251 Neu5Acα2-6Galβ-Sp8 119 252

252 Neu5Acα2-8Neu5Acα-Sρ8 1083 432

253 Neu5Acα2-8Neu5Acα2-3Galβl-4Glcβ-Sp0 732 219

254 Neu5Acβl-6GalNAcα-Sp8 2959 3196 255 Neu5Acb2-6Galbl-4GlcNAcb-Sρ8 38 11

256 Neu5Acβ2-6(Galβl-3)GalNAcα-Sp8 920 169

257 Neu5Gca2-3Galbl-3(Fucal-4)GlcNAcb-SpO 753 954

258 Neu5Gca2-3Galbl-3GlcNAcb-SpO 1069 -938

259 Neu5Gca2-3Galbl-4(Fucal-3)GlcNAcb-SpO 1194 294

260 Neu5Gcα2-3Galβl-4GlcNAcβ-SpO 907 -32

261 Neu5Gcα2-3Galβl-4Glcβ-SρO 353 736

262 Neu5Gcα2-6GalNAcα-Sp0 1772 1075

263 Neu5Gcα2-6Galβl-4GlcNAcβ-SpO s- 1175 475

264 Neu5Gcα-Sp8 179 199

Table 27. Determination of specific glycan binding by 7WC::His. Relative Fluorescence Units (RFU) detected at each glycan has been adjusted for specific and non-specific antibody background binding.

The specificity and degree of glycan binding of 7WC::His was influenced by pH where at pH5.0 the highest glycan binding was to [3OSO3][6OSO3]Galb1-4[6OSO3]GlcNAcb-Sp0 which was the same for both 6RG and 4WC, however, at pH 8.0 the highest glycan binding by 7WC::His occurred to various forms of glucose di and tri-saccharides (glycans 177-181 Table 27). These were not bound to any degree by 6RG::His, 4WC::His, 6RG::4WC::His or 4WC::6RG::His.

Generation of chimeras to alter their oligomeric status Chimeric apyrase oligomeric status and activities were determined by Size Exclusion Column chromatography and assaying and protein concentration of the collected fractions Mature 6RG forms an active monomer whereas mature 4WC forms an active oligomer (likely dimer); 4WC::6RG forms an active monomer whereas 6RG::4WC forms an active oligomer (Figure 78). These results demonstrate that we are able to modify active oligomeric status of recombinant soluble apyrases by generating chimeric apyrases.

General comparison of recombinant apyrase activities

A comparison of absolute specific activities was performed on insect cell expressed recombinant soluble 6RG, 4WC, 6RG::4WC and 4WC::6RG and E. coli expressed and refolded 7WC (either as the low activity mixed oligomeric status or the high activity monomer) (Table 28).

Table 28. Typical apyrase activities for insect cell expressed recombinant apyrases. ATP used as substrate.

Generation of diagnostic tools

We have demonstrated that the carbohydrate binding of each apyrase is specific and can be modified by altering the surrounding pH or by generating chimeras between the apyrases. These could be used to generate a variety of diagnostic tools , e.g., where either the apyrase is fixed to a solid surface or the apyrase is passed in solution over samples that are fixed to a solid surface. Detection of carbohydrate binding could then be achieved either by detection of bound apyrase, e.g., by using specific antibodies or by detection of apyrase activity.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims.

Claims

WHAT WE CLAIM IS:

1. A use for an apyrase or chimeric apyrase together with a substrate and a colour reaction mixture to indicate the presence or absence of a target carbohydrate.

2. A chimeric apyrase with altered carbohydrate binding compared to known native apyrases.

3. A chimeric apyrase gene encoding a chimeric apyrase having altered carbohydrate binding compared to known native apyrases.

4. A use of an apyrase or chimeric apyrase to remove or isolate a target carbohydrate from a liquid sample.

5. A use for nucleotide sequence information of at least two apyrases to construct a chimeric apyrase gene.

6. A method of using an apyrase or chimeric apyrase to specifically bind a target carbohydrate comprising the steps of:

b) removing said liquid from the apyrase or chimeric apyrase or vice versa.

7. A method of treating a disease ex-vivo using an apyrase or chimeric apyrase to specifically bind a target carbohydrate comprising the steps of:

b) removing said liquid from the apyrase or chimeric apyrase or vice versa.

8. An assay which includes an apyrase or chimeric apyrase which can specifically bind to a target carbohydrate.

9. A method for identifying the presence of a carbohydrate known to specifically bind to the carbohydrate of interest, comprising the steps of:

10. A substantially isolated nucleic acid molecule selected from the group consisting of:

a) the sequence shown in SEQ ID No. 1;

b) a complement of the sequence shown in SEQ ID No. 1 ;

c) a reverse complement to the sequences recited in a), and b); and

d) a reverse sequence to a sequence recited in c); and

11. A substantially isolated nucleic acid molecule selected from the group consisting of:

a) the sequence shown in SEQ ID No. 3;

b) a complement of the sequence shown in SEQ ID No. 3;

c) a reverse complement to the sequences recited in a), and b); and

d) a reverse sequence to a sequence recited in c); and

12. A substantially isolated nucleic acid molecule selected from the group consisting of: a) the sequence shown in SEQ ID No. 5;

b) a complement of the sequence shown in SEQ ID No. 5;

c) a reverse complement to the sequences recited in a), and b); and

d) a reverse sequence, to a sequence recited in c); and

13. A substantially isolated apyrase which has an amino acid sequence selected from the group consisting of:

a) SEQ ID No. 2; or

b) a functionally active fragment or variant of a) sequence recited in a).

14. A substantially isolated apyrase which has an amino acid sequence selected from the group consisting of:

a) SEQ ID No. 4; or

b) a functionally active fragment or variant of a) sequence recited in a).

15. A substantially isolated apyrase which has an amino acid sequence selected from the group consisting of:

a) SEQ ID No. 6; or

b) a functionally active fragment or variant of a) sequence recited in a).

16. An apyrase which has been substantially encoded by a nucleic acid molecule having a nucleotide sequence substantially as set forth in the group consisting of:

a) SEQ ID Nos. 1 , 3, and 5; or b) a complement of the sequence shown in a);

c) a reverse complement to the sequences recited in a), and b); and

d) a reverse sequence to a sequence recited in c); and

e) functionally active, fragments and variants of the sequences recited in a), b), c) and d).

17. An antibody which specifically binds to an apyrase having an amino acid sequence selected from the group consisting of:

a) SEQ ID Nos. 2, 4 or 6; or

b) a functionally active fragment or variant of a sequence recited in a).

18. The use of nucleotide sequence information of at least two apyrases to construct a chimeric apyrase gene having altered apyrase nucleotide substrate specificity, and/or apyrase activity and/or pH optimum with respect to one or more of the native apyrases from which the nucleotide sequence information was obtained

19. The use of nucleotide sequence information of at least two apyrases to construct a chimeric apyrase gene having altered phosphohydrolase activity with respect to one or more of the native apyrases from which the nucleotide sequence information was obtained.

20. The use as claimed in claim 18 wherein the altered phosphohydrolase activity narrows the nucleotide tri-phosphates on which the chimeric apyrase can act.

21. The use as claimed in claim 18 wherein the altered phosphohydrolase activity broadens the nucleotide tri-phosphates on which the chimeric apyrase can act.

22. The use of nucleotide sequence information of at least two apyrases to construct a chimeric apyrase gene having altered oligomeric status with respect to one or more of the native apyrases from which the nucleotide sequence information was obtained.

23. The use of nucleotide sequence information of at least two apyrases to construct a chimeric apyrase gene having altered carbohydrate specificity with respect to one or more of the native apyrases from which the nucleotide sequence information was obtained.

24. A transformed cell which includes a nucleic acid molecule selected from the group consisting of: , _*

a) SEQ ID No. 1 , 3, or 5; or

b) a complement of the sequence shown in a);

c) a reverse complement to the sequences recited in a), and b); and

d) a reverse sequence to a sequence recited in c); and

25. A host cell which includes a nucleic acid molecule selected from the group consisting of:

a) SEQ ID No. 1 , 3, or 5; or

b) a complement of the sequence shown in a);

, c) a reverse complement to the sequences recited in a), and b); and

d) a reverse sequence to a sequence recited in c); and

26. The use of a nucleic acid molecule selected from the group consisting of:

a) SEQ ID No. 1 , 3, or 5; or

b) a complement of the sequence shown in a);

e) functionally active fragments and variants of the sequences recited in a), b), c) and d)

as a probe.

27. A chimeric apyrase comprising a first fragment obtained from a first apyrase gene sequence and a second fragment obtained from a second apyrase gene sequence wherein said first and second apyrase gene sequences are either homologous, heterologous or isologous.

28. A chimeric apyrase as claimed in claim 27 wherein the first fragment is an 4N-terminus and the second fragment is a 1C-terminus.

29. A chimeric apyrase as claimed in claim 27 wherein the first and second fragments are conserved regions from different apryases.

30. A chimeric apyrase substantially as described herein with reference to any example and/or drawing thereof.

31. The use of a nucleic acid molecule as claimed in any one of claims 10, 11 , or 12 to identify apyrases from different organisms.

32. An oligonucleotide comprising at least 14-20 contiguous nucleotides selected from a nucleic acid molecule as claimed in any one claims 10, 11 , or 12.

33. A use of an oligonucleotide as claimed in claim 27 to identify or isolate an apyrase gene.

34. A host cell which includes a chimeric apyrase gene.

35. A transformed cell which includes a chimeric apyrase gene.

36. A chimeric apyrase gene encoding a chimeric apyrase as claimed in any one of claims 27 -30.

37. A polypeptide recombinantly produced from a nucleic acid as claimed in any one of claims 11 , 12 and 13. REFERENCES

Asian, K, Zhang J, Lakowicz JR, Geddes CD. (2004). Saccharide sensing using gold and silver nanoparticles-A review. J Fluoresence, 14:391-400

Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) "Basic local alignment search tool." J. MoI. Biol. 215:403-410.

Bendtsen, JD. Nielsen, H., von Heijne G., and Brunak S. (2004). Improved prediction of signal peptides: SignalP 3.0. J. MoI. Biol., 340:783-795. Chevillard, C, Cardenas, M.L., and Cornish-Bowden, A. (1993). The competition plot: a simple test of whether two reactions occur at the same active site. Biochem J 289 ( Pt 2), 599-604

Cohn JR, Uhm T, Ramu S, Nam YW, Kim DJ, Penmetsa RV, Wood TC, Denny RL, Young ND, Cook DR, and Stacey G.(2001). Differential regulation of a family of apyrase genes from Medicago truncatula. Plant Physiol. 125(4):2104-19.

Cuff JA, Barton GJ. (2000). Application of multiple sequence alignment profiles to improve protein secondary structure prediction. Proteins, 40(3):502-11.

Creighton (1984). Proteins. W. H. Freeman and Company

Emanuelsson, O., Nielsen, H., Brunak, S., and von Heijne, G. (2000). Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J. MoI. Biol., 300:1005-1016.

Etzler, M.E., Kalsi, G., Ewing, N.N., Roberts, N.J., Day, R.B., & Murphy, J.B. (1999) A Nod Factor Binding Lectin With Apyrase Activity On The Surface Of Legume Root Hairs. PNAS (USA) 96,

5856-5861.

Etzler, M. E. & Murphy, J.B. Nod factor binding protein from legume roots. United States Patent 6,465,716 October 15, 2002

Etzler, M. E. & Roberts, N.J. LNP, A Protein Involved in the Initiation of Mycorrhizal Infection of Plants. U.S. Patent Application No. 09/657,631. International Publication Number WO

02/20725 A2. 2001

Etzler, M.E. & Roberts, N.J. LNP, a protein involved in the initiation of mycorrhizal infection in plants. United States Patent 6,849,777 February 1, 2005

Fonseca FV, de Souza ALF, Mariano AC, Entringer PF, Gondim KC & Meyer-Fernandes JR. (2006). Trypanosoma rangelϊ. Characterization of a Mg-dependent ecto ATP- diphosphohydrolase activity. Experimental Parasitology, 112:76-84. Frohman MA, Dush MK, and Martin GR. (1988). Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 85(23):8998-9002.

Fromell K, Andersson M, Elihn K, CaId well KD. (2005). Nanoparticle decorated surfaces with potential use in glycosylation analysis. Colloids and Surfaces G: Biointerfaces 46: 84-91.

Grϋn CH, van Vliet SJ, Schiphorst WECM, Bank CMC, Meyer S, van Die I, van Kooyak Y. (2006). One-step biotinylation procedure for carbohydrates to study carbohydrate-protein interactions. Analytical Biochemistry 2006 on line.

Handa M, and Guidotti G. (1996). Purification and cloning of a soluble ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum). Biochem Biophys Res Commun.

218(3):916-23.

Ivanenkov W, Meller J, Kirley TL. (2005). Characterization of disulfide bonds in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): implications for NTPDase structural modeling. Biochemistry, 44(25):8998-9012.

Kalsi G, and Etzler ME. (2000). Localization of a Nod factor-binding protein in legume roots and factors influencing its distribution and expression. Plant Physiol. 124(3): 1039-48.

Kneller DG, Cohen FE, Langridge R. (1990). Improvements in protein secondary structure prediction by an enhanced neural network. J. MoI. Biol., 214(1 ):171 -82.

Kyte J, Doolittle RF. (1982). A simple method for displaying the hydropathic character of a protein.

J. MoI. Biol., 157(1 ):105-32.

Loh, EY., Elliott, JF., Cwirla, S, Lanier, LL., Davis, MM. (1989). Polymerase Chain Reaction with Single-Sided Specificity: Analysis of T Cell Receptor Sigma Chain. Science. 243(4888):217-221.

Manimala JC, Li Z, Jain A, VedBrat S, Gildersleeve JC. (2005). Carbohydrate array analysis of anti- Tn antibodies and lectins reveals unexpected specificities: Implications for diagnostic and vaccine development. ChemBioChem. 6:2229-2241.

Masarova J, Dey ES, Danielsson B. (2004). Optical lectin based biosensor as tool for bacterial identification. Polish J. Microbiology, 53, Suppl., 23-27.

McClelland, JL., and Rumelhart, DE. (1988) "Explorations in Parallel Distributed Processing" vol 3. pp 318-362. MIT Press, Cambridge MA. Ohara, O. Dorit, RL. and Gilbert, W. (1989). One-Sided Polymerase Chain Reaction: The Amplification of cDNA. PNAS USA. 86: 5673-5677

Rϋdiger H, Gabius, H-J. (2001). Plant lectins: Occurrence, biochemistry, functions and applications. Glycoconjugate Journal 18: 589-613.

Sambrook, J., Fritsch, EF., and Maniatis, T. (1989). Molecular Cloning : A Laboratory Manual (3 Volume Set). Cold Spring Harbor Laboratory Press, ISBN: 0879693096.

Roberts, N.J., Brigham, J.T., Wu, B., Murphy, J.B. Volpin, H.D., Phillips, DA, & Etzler, M.E.

(1999) A Nod factor binding lectin is a member of a distinct class of apyrases that may be unique to the legumes. Molecular and General Genetics 262, 261-267.

Vasconcelos EG, Ferreira ST, de Carvalho TMU, de Souza W, Kettlun AM, Mancila M, Valenzuela MA, Verjovski-Almeida S. (1996). Partial purification and immunohistochemical localization of ATP diphosphohydrolase from Schistomsoma mansoni. J. B.C., 271:22139-22145.

Wu, JJ, Choi LE, and Guidotti G. (2005). N-linked oligosaccharides affect the enzymic activity of CD39: Diverse interactions between seven N-linked glycosylation sites. Molecular Biology of the Cell. 16:1661-1672.

Zimmermann H, Braun N, Kegel B, Heine P. (1998). New insights into molecular structure and function of ecto-nucleotidases in the nervous system. Neurochem. Int. 32:421-425.

Choreo and Goodman, Ace. Chem. REs., 1993 26 266-273

Freidinger, R.M., Perlow, D.S., Veber, D.F., J. Org. Chem. 1982, 59, 104-109

Gallop, M.A., Barrett, R.W., Dower, W.J., Fodor, S.P.A. and Hogan, Jr., J.C. Nature Biotechnology, 1997 15 328-330.

Olson et al., J. Med. Chem., 1993 36 3039-3049.

Smythe, M.L., von Itzstein, M., J. Am. Chem. Soc. 1994, 116, 2725-2733

Creighton, 1984, Proteins, W. H. Freeman and Company