NZ622172B2 - Endoglycosidase from streptococcus pyogenes and methods using it - Google Patents
Endoglycosidase from streptococcus pyogenes and methods using it Download PDFInfo
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- NZ622172B2 NZ622172B2 NZ622172A NZ62217212A NZ622172B2 NZ 622172 B2 NZ622172 B2 NZ 622172B2 NZ 622172 A NZ622172 A NZ 622172A NZ 62217212 A NZ62217212 A NZ 62217212A NZ 622172 B2 NZ622172 B2 NZ 622172B2
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl β-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 230000002147 killing Effects 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 101710017890 large T Proteins 0.000 description 1
- 101710030587 ligN Proteins 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 101700077585 ligd Proteins 0.000 description 1
- 230000028744 lysogeny Effects 0.000 description 1
- 230000002101 lytic Effects 0.000 description 1
- 210000004962 mammalian cells Anatomy 0.000 description 1
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic Effects 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 230000003535 nephritogenic Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000001915 proofreading Effects 0.000 description 1
- 230000001902 propagating Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 210000001519 tissues Anatomy 0.000 description 1
- 238000004450 types of analysis Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
- C07K2317/41—Glycosylation, sialylation, or fucosylation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/005—Glycopeptides, glycoproteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01096—Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (3.2.1.96)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56944—Streptococcus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Abstract
Disclosed is an isolated polypeptide comprising (a) the amino acid sequence of SEQ ID NO: 1; (b) a variant thereof having at least 95% identity to the amino acid sequence of SEQ ID NO: 1 over at least 810 contiguous amino acids of SEQ ID NO: 1 and having the endoglycosidase activity of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. Also disclosed is an isolated polypeptide capable of binding to IgG and which does not have endoglycosidase activity comprising (a) the amino acid sequence of SEQ ID NO: 2; (b) a variant thereof having at least 95% identity to the amino acid sequence of SEQ ID NO: 1 over at least 810 contiguous amino acids of SEQ ID NO: 1, in which the amino acid equivalent to glutamic acid at position 186 is substituted. de consisting of the amino acid sequence of SEQ ID NO: 1. Also disclosed is an isolated polypeptide capable of binding to IgG and which does not have endoglycosidase activity comprising (a) the amino acid sequence of SEQ ID NO: 2; (b) a variant thereof having at least 95% identity to the amino acid sequence of SEQ ID NO: 1 over at least 810 contiguous amino acids of SEQ ID NO: 1, in which the amino acid equivalent to glutamic acid at position 186 is substituted.
Description
ENDOGLYCOSIDASE FROM STREPTOCOCCUS PYOGENES AND METHODS USING |T
Field of the Invention
The present invention relates to a novel endoglycosidase, mutants f
lacking glycan hydrolyzing activity, and its use in methods of hydro lyzing the glycan
of glycoproteins.
ound of the Invention
Endoglycosidase S (EndoS) is secreted by a number of serotypes of
Streptococcus pyogenes and has a specific endoglycosidase activity on native IgG by
hydrolyzing the conserved glycans attached to the asparagine 297 residue on the
heavy chains of IgG, Collin and Olsen, The EMBO Journal, 2001, 20 055.
EndoS is the first known bacterial enzyme with a unique specificity for native IgG. In
contrast, the activities of other known endoglycosidases require or are enhanced by
denaturation of the rotein substrate.
dies such as IgG have many applications in basic ch as well as in
diagnostics and drug development. In some of these applications, such as
immunohistochemistry, immunoassays, tumour detection, radiotherapy,
crystallographic studies of antibody binding sites and immunotargeting, it is more
convenient to use Fab nts than whole IgG molecules. Some of the advantages
ofusing Fab nts are that they will not be affected by Fc receptors on cells or
precipitate antigen, they display a d immunogenicity and are less susceptible to
phagocytosis, and that radio labelled Fab fragments are more rapidly cleared from
tissue than whole IgG molecules. For other applications, it is desirable to use Fc
fragments of IgG. In further applications, it may be desirable to use deglycosylated
versions of the antibodies or other glycoproteins.
The cleavage of IgG into Fab and PC fragments is most often carried out using
lytic enzymes such as pepsin or papain. These enzymes often cleave other
proteins, so the cleavage reaction generally has to be performed on a purified IgG
fraction. Furthermore, pepsin and papain typically cleave IgG in more than one place.
This means that the fragments ed often do not correspond to whole Fab or PC
nts, and even if cleavage does result in Fab and PC fragments, they are
typically susceptible to further cleavage into smaller fragments. The isolation of PC
fragments from Fab fragments is most often carried out using protein A or G affinity
separation columns, which utilise the Fc-binding properties of the bacterial proteins A
and G.
Many ent glycoproteins have utility in therapeutic applications. Methods
to analyse the glycosylation of such proteins have utility in the research and
development ofthe proteins as therapeutics. It may also be desirable to provide
osylated versions of these proteins.
Summary of the Invention
The inventors have identified a novel endoglycosidase from serotype M49
Streptococcus pyogenes, referred to herein as 9. EndoS49 was isolated from
strain NZl3 l a nephritogenic and highly transformable strain of serotype M49.
NZl3l strain is a clinical isolate from a case of acute treptococcal
glomerulonephritis in New Zealand. At a protein level, EndoS49 has less than 40%
identity to EndoS, and is a 90kDa protein, compared to the lO8kDa of EndoS.
9 has deglycosylation activity for a r range of proteins than EndoS.
The enzyme is a 90kDa enzyme, having a family 18 glycoside hydrolase
catalytic domain. EndoS49 hydrolyzes glycan on human glycoproteins, and in
particular IgGl-4, and alpha-l-microglobulin. EndoS49 can be used in the hydrolysis
of s on human glycoproteins including IgG and alpha-l-microglobulin.
EndoS49 can thus be used in glycoprofiling analysis in which the enzyme is contacted
with a glycoprotein, and the products ed are separated for analysis of the
glycans and the protein. EndoS49 can also be used to prepare deglycosylated
proteins. The enzyme can be modified to reduce or remove endoglycosidase activity.
The modified EndoS49 polypeptide which lacks endoglycosidase activity can
be used in methods for isolating glycosylated and/or functionally active IgG. By
using such a modified EndoS49 polypeptide in ation with an additional IgG-
binding reagent which is capable of binding denatured and/or deglycosylated IgG, the
inventors have also fied a method for assessing the glycosylation state or
onal quality of an IgG-containing sample.
In accordance with the present invention, there is thus provided a ptide
comprising
(a) the amino acid sequence of SEQ ID NO: 1 ;
(b) a variant thereof having at least 50% identity to the amino acid
sequence of SEQ ID NO: 1 and having endogycosidase activity; or
(c) a fragment of either thereof having endoglycosidase activity.
The invention also provides a polypeptide comprising
(a) the amino acid ce of SEQ ID NO: 1;
(b) a variant thereof having at least 95% identity to the amino acid
sequence of SEQ ID NO: 1 over at least 810 contiguous amino acids of SEQ
ID NO: 1 and having the endoglycosidase activity of a polypeptide consisting
of the amino acid sequence of SEQ ID NO: 1.
The invention also provides a ptide capable of binding to IgG and
which does not have ycosidase activity comprising
(a) the amino acid sequence of SEQ ID NO: 2;
(b) a variant f having at least 50% identity to the amino acid
sequence of SEQ ID NO: 1, in which the amino acid equivalent to glutamic
acid at position 186 is substituted; or
(c) a fragment of either thereof.
The ion further provides a polypeptide capable of binding to IgG and
which does not have endoglycosidase activity comprising
(a) the amino acid sequence of SEQ ID NO: 2;
(b) a variant thereof having at least 95% identity to the amino acid
sequence of SEQ ID NO: 1 over at least 810 contiguous amino acids of SEQ
ID NO: 1, in which the amino acid equivalent to ic acid at position 186
is substituted.
The invention also provides polynucleotides, expression vectors and host cells
encoding or expressing the polypeptides of the invention. The invention also relates
to the use of the polypeptides of the invention in a method of determining or ing
the glycosylation state of the protein, and in particular of an antibody, in particular an
IgG antibody.
The ion also provides a method for isolating IgG from an IgG-
containing sample, which method comprises:
(a) contacting said IgG-containing sample with a modified EndoS49
polypeptide which lacks IgG endoglycosidase activity
(b) separating said EndoS49 from the contacted sample;
y obtaining isolated IgG.
(followed by page 3a)
The invention further provides a method for assessing the glycosylation status
of a glycoprotein with a polypeptide comprising incubating a glycoprotein with a
polypeptide comprising:
(a) the amino acid sequence of SEQ ID NO: 1;
(b) a variant thereof having at least 95% identity to the amino acid
sequence of SEQ ID NO: 1 over at least 810 uous amino acids of SEQ
ID NO: 1 and having the endoglycosidase activity of a polypeptide consisting
of the amino acid sequence of SEQ ID NO: 1.
In one example, the method comprises assessing the glycosylation status of a
rotein comprising ting a glycoprotein with a polypeptide according to
the present invention, and analysing the products produced.
Additionally there is provided method of assessing the ylation state or
functional quality of an IgG-containing sample, which method comprises taking a first
and a second mple of the IgG-containing sample, and wherein steps (a) and (b)
according to the method above are applied to the first sub-sample, and wherein steps
(a) and (b) as above are applied to the second sub-sample except the EndoS49
polypeptide is substituted with an alternative IgG-binding reagent which is capable of
binding denatured and/or deglycosylated IgG, and further comprising:
(c) quantifying the amount of IgG bound to the 9 polypeptide in
the first sub-sample, and the amount of IgG bound to the alternative IgG-
binding reagent in the second sub-sample; and
(d) comparing both the amounts of bound IgG determined in (c);
and thereby assessing the ylation state or functional y of an IgG
containing sample.
The modified enzyme of the present invention may also be used in methods
for ing Fab or Fc fragments of IgG. The methods of the invention make use of a
(followed by page 4)
highly specific IgG cleaving enzyme from S. pyogenes, IdeS oglobulin G-
degrading enzyme of §. pyogenes), and an EndoS49 polypeptide.
In one method of the invention, a sample containing IgG is contacted with
IdeS and an EndoS49 polypeptide, which is a modified EndoS49 polypeptide which
lacks endoglycosidase ty as described above.
In the methods of the invention, lly IdeS cleaves the IgG into Fab and Fe
fragments and the EndoS49 polypeptide binds to the Fc fragments. The PC fragments
are then separated from the Fab fragments.
This method is particularly useful for isolating Fab or PC fragments from
samples comprising purified IgG. More specifically, it is useful for ing Fab or
PC fragments from a sample comprising IgG purified using the modified EndoS49
polypeptide of the invention. However, the method can also be d for use on
samples containing unpurified IgG, such as serum, cell lysate or cell culture medium.
Also provided are kits for carrying out the methods according to the invention.
Brief Description of the Figures
Figure 1. lW alignment of EndoS49 and EndoS reveals two different
proteins. 9 and EndoS was aligned using ClustalW in the software MacVector.
GHl8 catalytic motif (D**D*D*E) is present at position 179-186 with Glul86 as the
catalytic residue.
Figure 2. EndoS49 has activity on roteins. A. 1 ug of EndoS49, its
catalytic mutant and the truncated versions was incubated with 3 ug human IgG in
PBS overnight at 37°C and ed on a SDS-PAGE gel and with LCAlectin blot. B.
1 ug EndoS49 and 9(El86L) was incubated with 3 ug of sses 1-4 of
human IgG and analyzed as above. C. 1 ug EndoS49 and its mutants were incubated
with 3 ug Alpha-l-microglobulin and analyzed on a 10 % SDS-PAGE gel.
Figure 3. EndoS49 binds to IgG. 4, 2 and 1 ug of EndoS49 and its mutants
were immobilized on a PVDF membrane and ted with human IgG and later
with protein-G coupled to HRP.
Figure 4. The genomic context of nd0S49 and ndoS. A comparison of the
genes surrounding nd0S49 and ndoSin GAS strains NZl3l (M49) and 5005 (M1) was
carried out in MacVector.
Figure 5. Phylogenetic analysis of EndoS49 and other bacterial
endoglycosidases.
Figure 6. SDS Page gel of Avastin and Erbitux after digestion with EndoS or
EndoS49 followed by IdeS digestion.
Brief Description of the Seguences
SEQ ID NO: 1 is an amino acid sequence of an EndoS49 polypeptide ed
from S. es M49 pe NZl3 l.
SEQ ID NO: 2 is an amino acid sequence of a modified EndoS49 polypeptide
(El86L) derived from the sequence of SEQ ID NO: 1.
SEQ ID NO: 3 is a nucleotide sequence encoding 9 polypeptide
SEQ ID NO: 4 is an amino acid sequence of IdeS isolated from S. pyogenes
APl.
Detailed Description of the Invention
lpolypeptidefeatures
The present invention relates to a novel polypeptide 9. The invention
also provides various methods which utilize the bacterial proteins EndoS49 and IdeS,
as well as other proteins. The terms protein, peptide and polypeptide are used
interchangeably herein. It will be understood that certain polypeptides and methods
ofthe invention require an EndoS49 polypeptide having endoglycosidase activity,
whereas other polypeptides and s of the invention require a modified EndoS49
polypeptide lacking said activity.
The following section relates to general features of all polypeptides of the
invention, and in particular to variations, alterations, modifications or derivatisations
ofamino acid sequence which are included within the polypeptides ofthe invention.
It will be understood that such variations, alterations, modifications or derivatisations
ofpolypeptides as are described herein are subject to the requirement that the
polypeptides retain any further required activity or characteristic as may be specified
subsequent sections of this disclosure.
Variants of polypeptides of the invention may be defined by particular levels
ofamino acid identity which are described in more detail in subsequent sections of
this disclosure. Amino acid identity may be ated using any suitable thm.
For example the PILEUP and BLAST thms can be used to calculate homology
or line up ces (such as identifying equivalent or corresponding sequences
ally on their t settings), for example as described in Altschul S. F. (1993) J
Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215 :403-10. Software
for performing BLAST analyses is publicly available through the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves
first identifying high scoring sequence pair (HSPs) by identifying short words of
length W in the query sequence that either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a se
sequence. T is referred to as the ourhood word score old (Altschul et al,
supra). These initial neighbourhood word hits act as seeds for initiating searches to
find HSPs containing them. The word hits are extended in both directions along each
sequence for as far as the cumulative alignment score can be increased. Extensions
for the word hits in each direction are halted when: the tive alignment score
falls off by the quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more negative-scoring
residue alignments; or the end of either sequence is reached. The BLAST algorithm
parameters W, T and X determine the sensitiVity and speed of the alignment. The
BLAST program uses as ts a word length (W) of 11, the BLOSUM62 scoring
matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-
10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of
both strands.
The BLAST algorithm performs a statistical analysis of the similarity between
two ces; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:
5873-5787. One measure of similarity provided by the BLAST algorithm is the
smallest sum ility (P(N)), which provides an indication of the probability by
which a match between two polynucleotide or amino acid sequences would occur by
chance. For example, a sequence is considered similar to another sequence if the
smallest sum probability in comparison of the first sequence to the second sequence is
less than about 1, preferably less than about 0.1, more preferably less than about 0.01,
and most preferably less than about 0.001. Alternatively, the UWGCG Package
provides the T program which can be used to calculate homology (for
example used on its t settings) eux et al (1984) Nucleic Acids Research
12, 5).
It will be understood that variants of polypeptides ofthe invention also
includes substitution ts. tution variants preferably involve the replacement
of one or more amino acids with the same number of amino acids and making
2012/067841
conservative amino acid substitutions. For example, an amino acid may be
substituted With an alternative amino acid having similar properties, for example,
another basic amino acid, another acidic amino acid, another neutral amino acid,
another charged amino acid, another hydrophilic amino acid, another hydrophobic
amino acid, another polar amino acid, another aromatic amino acid or another
aliphatic amino acid. Some properties of the 20 main amino acids Which can be used
to select le substituents are as follows:
Ala aliphatic, hydrophobic, neutral Met hydrophobic, neutral
Cys polar, hydrophobic, neutral Asn polar, hilic, neutral
Asp polar, hydrophilic, charged (-) Pro hydrophobic, neutral
Glu polar, hydrophilic, d (-) Gln polar, hilic, neutral
Phe aromatic, hydrophobic, neutral Arg polar, hydrophilic, charged (+)
Gly aliphatic, neutral Ser polar, hydrophilic, neutral
His aromatic, polar, hydrophilic, Thr polar, hydrophilic, neutral
charged (+)
Ile aliphatic, hydrophobic, neutral Val aliphatic, hydrophobic, neutral
Lys polar, hydrophilic, d(+) Trp aromatic, hydrophobic, neutral
Leu aliphatic, hydrophobic, neutral Tyr aromatic, polar, hydrophobic
The polypeptides of the invention and for use in the invention may be in a
substantially isolated form. It Will be understood that the polypeptide may be mixed
With carriers or diluents Which Will not interfere With the intended purpose of the
polypeptide and still be regarded as substantially ed. A ptide for use in
the invention may also be in a substantially purified form, in Which case it Will
generally comprise the polypeptide in a preparation in Which more than 50%, e.g.
more than 80%, 90%, 95% or 99%, by weight of the polypeptide in the preparation is
a polypeptide of the ion.
The amino acid sequence of polypeptides of the invention and for use in the
invention may be d to include non-naturally occurring amino acids or to
increase the stability of the compound. When the polypeptides are ed by
tic means, such amino acids may be introduced during production. The
polypeptides may also be modified following either synthetic or recombinant
production.
Polypeptides of the invention or for use in the invention may also be produced
using o acids. In such cases the amino acids Will be linked in reverse sequence
in the C to N orientation. This is conventional in the art for producing such
polypeptides.
A number of side chain modifications are known in the art and may be made
to the side chains of the ptides, subject to the polypeptides retaining any r
required ty or characteristic as may be specified herein.
It Will also be understood that the polypeptides of the invention and used in the
ion may be chemically modified, e. g. post-translationally modified. For
example, they may be glycosylated, phosphorylated or comprise modified amino acid
residues. They may be modified by the addition of a signal sequence to promote
insertion into the cell membrane.
The polypeptides of the invention may also be tised or modified to assist
With their isolation or ation. Thus, in one embodiment of the ion, the
polypeptide for use in the invention is derivatised or modified by addition of a ligand
Which is capable of binding directly and specifically to a separation means.
Alternatively, the polypeptide is derivatised or d by addition of one member of
a binding pair and the separation means comprises a reagent that is derivatised or
modified by addition ofthe other member of a binding pair. Any le binding pair
can be used. In a preferred embodiment Where the polypeptide for use in the
invention is derivatised or modified by on of one member of a binding pair, the
polypeptide is preferably histidine-tagged or biotin-tagged. Typically the amino acid
coding ce of the histidine or biotin tag is included at the gene level and the
proteins are expressed recombinantly in E. coli. The histidine or biotin tag is typically
present at one end of the polypeptide, either at the N—terminus or at the C-terminus.
The histidine tag typically consists of six histidine residues, although it can be longer
than this, typically up to 7, 8, 9, 10 or 20 amino acids or shorter, for example 5, 4, 3, 2
or 1 amino acids. Furthermore, the histidine tag may contain one or more amino acid
substitutions, preferably conservative substitutions as defined above.
EndoS49 polypeptides having endoglycosidase activity
The 9 polypeptide in this instance is preferably S. pyogenes 9,
or a variant or fragment of S. pyogenes EndoS49 which retains endoglycosidase
activity. The variant may be an EndoS49 polypeptide from r Streptococcus
equi, Streptococcus zooepidemicus or, ably, Streptococcus pyogenes
The EndoS49 polypeptide may comprise:
(a) the amino acid sequence of SEQ ID NO: 1 ;
(b) a variant thereof having at least 50% identity to the amino acid
sequence of SEQ ID NO: 1 and having ycosidase activity; or
(c) a fragment of either thereof having endoglycosidase activity.
Preferably, the polypeptide comprises, or consists of, the ce of SEQ ID
NO: 1. SEQ ID NO: 1 is the sequence of EndoS49 from S. pyogenes. The EndoS49
polypeptide of the invention may onally not comprise a signal sequence.
Variant polypeptides as described in this section are those for which the amino
acid sequence varies from that in SEQ ID NO: 1, but which retain the
endoglycosidase activity ofEndoS49. Such variants may include allelic variants and
the deletion, ation or addition of single amino acids or groups of amino acids
within the n sequence, as long as the peptide maintains IgG endoglycosidase
activity.
The variant sequences typically differ by at least 1, 2, 3, 5, 10, 20, 30, 50, 100
or more mutations (which may be substitutions, deletions or insertions of amino
. For example, from 1 to 100, 2 to 50, 3 to 30 or 5 to 20 amino acid
substitutions, deletions or insertions may be made, provided the modified polypeptide
retains activity as an IgG-specific endoglycosidase.
Variants of the amino acid sequence of SEQ ID NO: 1 preferably contain
residues 179 to 186 of SEQ ID NO: 1, and in particular include the motif D**D*D*E.
These amino acids constitute a family 18 glycoside hydrolase tic domain. The
glutamic acid at position 186 is essential for enzymatic activity. Most preferably,
therefore, the variant of SEQ ID NO: 1 ns glutamic acid at the position
lent to position 186 of SEQ ID NO: I. The variant of SEQ ID NO: I may
contain residues 179 to 186 of SEQ ID NO: 1 having one or more conservative
substitutions, provided that the variant contains glutamic acid at the position
equivalent to position 186 of SEQ ID NO: 1.
2012/067841
lly, polypeptides which display the endoglycosidase activity of
EndoS49 with more than about 50%, 55% or 65% identity, preferably at least 70%, at
least 80%, at least 90% and particularly preferably at least 95%, at least 97% or at
least 99% identity, with the amino acid sequence of SEQ ID NO: 1 are considered
ts of the n The identity ofvariants of SEQ ID NO: 1 may be measured
over a region of at least 100, at least 250, at least 500, at least 750, at least 800, at
least 810, at least 820, at least 930, at least 940 or more contiguous amino acids of the
sequence shown in SEQ ID NO: 1, or more preferably over the full length of SEQ ID
NO: 1.
The fragment of the EndoS49 polypeptide used in the invention is typically at
least 400, 500, 600, 700, 750, 800, or 825 amino acids in length, as long as it retains
the IgG endoglycosidase ty of EndoS. Preferably, the fragment of the EndoS49
polypeptide used in the invention encompasses residues 179 to 186 of SEQ ID NO: 1.
Polypeptides for use in the present invention may be isolated from any suitable
organism that expresses an EndoS49 polypeptide or a variant of an EndoS49
polypeptide. Typically, the EndoS49 polypeptide is isolated from suitable EndoS49
expressing strains of Streptococcus, preferably strains of S. pyogenes, and in
ular those of pe M49.
Isolation and purification of 9 from an expressing S. pyogenes e,
or from cultures of other cells expressing 9 is typically on the basis of
endoglycosidase activity. Preferably the purification method involves an ammonium
sulphate precipitation step and an ion exchange tography step. According to
one method, the culture medium is fractionated by adding increasing amounts of
ammonium sulphate. The amounts of ammonium sulphate may be 10 to 80%.
Preferably the culture medium is fractionated with 50% ammonium sulphate, and the
resulting supernatant is further precipitated with 70% ammonium sulphate. Pelleted
polypeptides may then be subjected to ion exchange chromatography, for example by
FPLC on a Mono Q column. Eluted ons may be d for endoglycosidase
activity and peak activity fractions may be pooled. Fractions may be analysed by SDS
PAGE. Fractions may be stored at -80°C.
Polypeptides for use in the invention may also be prepared as fragments of
such isolated polypeptides. Further, the EndoS49 polypeptides may also be made
synthetically or by recombinant means. For example, a recombinant EndoS49
polypeptide may be produced by transfecting ian cells in culture with an
expression vector comprising a nucleotide sequence encoding the polypeptide
operably linked to suitable control ces, culturing the cells, extracting and
ing the 9 polypeptide produced by the cells.
The EndoS49 polypeptides of invention described in this n display
endoglycosidase activity. Preferably, the polypeptide hydrolyses IgG or IgG Fc
fragments by hydrolysing glycan linked of a full-length IgG heavy chain polypeptide.
Preferably the EndoS49 polypeptide of the invention also has endoglycosidase
activity, and is capable of glycan hydrolysis of l-microglobulin.
The endoglycosidase activity may be determined by means of a suitable assay.
For example, a test polypeptide may be incubated with glycoprotein such as IgG or
alpha-l-microglobulin at a suitable temperature, such as 37°C. The starting materials
and the reaction products may then be analysed by SDS PAGE. Typically, the
molecular mass of the IgG heavy chain is reduced by approximately 3kDa to 4kDa if
the test polypeptide has IgG endoglycosidase ty. Another assay for determining
r a test polypeptide has IgG endoglycosidase ty is by detection of
glycosylated IgG using Lens culinarz’s agglutinin lectin (LCA), optionally using
horseradish peroxidase and peroxidase ate. Typically, the carbohydrate signal is
d if the test polypeptide has IgG endoglycosidase activity. Another assay for
determining whether a test polypeptide has IgG endoglycosidase activity is by
incubation of a test polypeptide with purified IgG Fc fragments followed by reduction
ofthe sample with 10 mM dithiotreitol and mass spectroscopy (MALDI-TOF)
analysis. Endoglycosidase activity can also be measured for EndoS49 polypeptides
by using alpha-l-microglobulin in place of IgG in the assays mentioned above.
The ycosidase activity of the polypeptides can be further characterised
by inhibition studies.
The EndoS49 polypeptide is capable of hydrolyzing glycoprotein molecules
t in a sample taken from a subject. Thus, where the subject is a human, the
EndoS49 polypeptide is capable of hydrolyzing the glycans in glycoproteins of a
subject, such as on the heavy chains of human IgG or alpha-l-microglobulin.
EndoS49 is capable of hydrolyzing human IgG of all four subclasses 4). In
preferred embodiments, the EndoS49 polypeptide has the ability to hydrolyze human
IgG and alphamicroglobulin.
EndoS49 polypeptides which lack endoglycosidase activity
The EndoS49 polypeptide in this instance may also be modified S. es
EndoS49, which has been engineered to lack endoglycosidase activity but which
possesses IgG binding activity. Such modified 9 is particularly useful in the
methods described herein. By IgG binding ty it will be understood that the
modified EndoS49 binds to IgG, or a variant or fragment thereof, in particular the Fc
fragment thereof, which is normally ylated. By “normally glycosylated” it will
be understood that the IgG molecule, or variant of fragment thereof, is a glycoprotein
comprising at least the IgG polypeptide heavy chain (or variant of fragment thereof)
d to at least one carbohydrate group as found coupled to lly occurring
IgG molecules. In particular, the at least one carbohydrate group is a glycan linked to
the asparagine residue corresponding to residue 297 of a full-length IgG heavy chain
polypeptide.
The EndoS49 ptide is preferably engineered by irected
mutagenesis. By IgG g activity it will be understood that the EndoS49
polypeptides described in this n bind at least one, preferably two, three or all
four of the IgG subclasses, IgG1_4. Preferably the at least one IgG subclass is bound
with high affinity and/or high specificity.
By high affinity it is meant that the binding affinity constant (KD) for the
interaction of the modified EndoS49 with an IgG subclass is greater than 0.05 uM,
preferably greater than 0.06 uM, 0.07 uM or 0.08 uM. Binding activity may be
determined, and binding affinity may be assessed by any suitable means. For
example, by surface plasmon resonance interaction is, brium dialysis
analysis, or any standard biochemical methods in conjunction with, for example,
Scatchard analysis.
The variant may be derived from an EndoS49 polypeptide from another
organism, such as another bacterium, as is described in the preceding section with the
exception that the t in this instance lacks endoglycosidase activity but possesses
IgG binding activity. The d EndoS49 polypeptide may comprise:
(a) the amino acid sequence of SEQ ID NO: 2;
(b) a variant f having at least 50% identity to the amino acid
sequence of SEQ ID NO: 2 which lacks endoglycosidase activity; or
(c) a fragment of either thereof which lacks endoglycosidase activity.
Preferably, the polypeptide comprises, or consists of, the sequence of SEQ ID
NO: 2. SEQ ID NO: 2 is derived from the sequence of SEQ ID NO: 1, but has been
engineered to lack endoglycosidase activity by the substitution of glutamic acid (E)
for leucine (L) at position 186 of SEQ ID NO: 1. Such polypeptides typically possess
IgG-binding activity as described above.
Variant polypeptides as described in this n are those for Which the amino
acid sequence varies from that in SEQ ID NO: 2, but Which lack endoglycosidase
activity and retain IgG-binding ty. Such variants may include allelic ts
and the deletion, cation or addition of single amino acids or groups of amino
acids Within the protein sequence, as long as the peptide maintains the above
characteristics.
The variant sequences typically differ by at least 1, 2, 3, 5, 10, 20, 30, 50, 100
or more ons (Which may be substitutions, deletions or insertions of amino
acids). For example, from 1 to 100, 2 to 50, 3 to 30 or 5 to 20 amino acid
substitutions, deletions or insertions may be made, provided the modified polypeptide
lacks endoglycosidase activity and retains IgG-binding activity.
Typically, polypeptides Which lack endoglycosidase activity and retain IgG-
binding activity With more than about 50%, 55% or 65% identity, preferably at least
70%, at least 80%, at least 90% and particularly preferably at least 95%, at least 97%
or at least 99% identity, With the amino acid sequence of SEQ ID NO: 2 are
considered variants of the protein The identity ofvariants of SEQ ID NO: 2 may be
measured over a region of at least 100, at least 250, at least 500, at least 750, at least
800, at least 820, at least 830 or more contiguous amino acids of the sequence shown
in SEQ ID NO: 2, or more preferably over the full length of SEQ ID NO: 2.
The fragment of the EndoS49 ptide used in the invention is lly at
least 300, 400, 500, 600, 700, 750, 800 or 830 amino acids in length, as long as it
lacks endoglycosidase activity and retains IgG-binding activity.
In an alternative , an EndoS49 protein With the desired characteristics
can be produced by altering a nucleotide encoding an EndoS49 protein, and then
expressing said tide in a le system. Suitable methods include site-
directed mutagenesis of the nucleotide encoding the protein. This technique has been
Widely used in the study of protein functions. The technique is typically
ucleotide-based and involves the following steps:
WO 37824
(l) Cloning the DNA encoding the protein of interest into a plasmid
vector.
(2) Denaturing the plasmid DNA to produce single strands.
(3) ting the denatured DNA with a tic oligonucleotide (or
oligonucleotides) complementary to the target sequence but orating the desired
on(s) (point mutation, deletion, or insertion), such that the tic
oligonucleotide anneals to the target .
(4) Extending the mutated strand by a DNA-polymerase using the plasmid
DNA strand as the template.
(5) Propagating the heteroduplex (mutated/non-mutated ) by
transformation in E. 6011'.
After propagation, about 50% of the produced heteroduplexes are mutants and
the other 50% are "wild type" (no mutation). Selection and enrichment s are
used to favor the production of mutants. For example, the parental non-mutated
strand can be digested with a restriction enzyme that only digests methylated DNA
(DpnI). This allows removal ofthe parental strand from the reaction before
transformation ofE. coli by since the newly synthesized strands are un-methylated
while the parental strand (if purified from the correct E. 6011' background) is
methylated.
Alternatives to site-directed mutagenesis include:
(1) Polymerase chain reaction (PCR) based methods using specific
mutagenic primers, or error-prone PCR with subsequent screening for desired
mutations or loss/gain of protein function.
(2) Introduction of a plasmid harboring the gene of interest into an E. coli
mutator strain (deficient in DNA proofreading systems) and subsequent screening for
desired mutations or loss/gain of protein function.
(3) Chemical synthesis of l or whole genes containing the desired
mutations and subsequent uction into an appropriate protein expression system.
Alternatively, an EndoS49 protein with the desired characteristics can be
produced by dependent methods, which include chemical synthesis of parts of
a polypeptide with the desired mutation.
Polypeptides for use in the invention may also be prepared as fragments of
such ed polypeptides. Further, the EndoS49 polypeptides may also be made
synthetically or by recombinant means. For example, a recombinant EndoS49
polypeptide may be produced by transfecting mammalian cells in culture with an
expression vector sing a nucleotide sequence encoding the polypeptide
operably linked to suitable control sequences, culturing the cells, extracting and
purifying the EndoS49 polypeptide produced by the cells.
The EndoS49 polypeptide is capable of binding to IgG molecules present in a
sample taken from a subject. Thus, where the subject is a human, the EndoS49
ptide is capable of binding human IgG. EndoS49 is capable of g human
IgG of all four subclasses (IgG1_4).
Polynucelotides, vectors and host cells
The invention also relates to polynucleotides ng the above polypeptides,
and their use in medicine. In particular the invention relates to polynucleotides
comprising or consisting of (a) the coding sequence of SEQ ID N03 or a
complementary sequence thereto; (b) sequence which hybridises under stringent
conditions to the sequences defined in (a); (c) sequence which is degenerate as a result
ofthe genetic code to sequence as defined in (a) or (b); (d) sequence having at least
60% identity to sequences defined in (a) (b) or (c); and (e) fragments of the above
ces.
Typically the polynucleotide is DNA. However, the invention may
comprise RNA cleotides. The polynucleotides may be single or double
ed, and may include within them synthetic or modified nucleotides.
A cleotide of the invention can hybridize to the coding sequence or the
ment of the coding sequence of SEQ ID NO: 3 at a level cantly above
ound. Background hybridization may occur, for example, because of other
DNAs present in a DNA library. The signal level generated by the ction
between a polynucleotide of the invention and the coding sequence or complement of
the coding sequence of SEQ ID NO: 3 is typically at least 10 fold, preferably at least
100 fold, as intense as interactions between other polynucleotides and the coding
sequence of SEQ ID NO: 3. The intensity of interaction may be measured, for
example, by radiolabelling the probe, e. g. with 32P. Selective hybridisation may
typically be achieved using conditions of medium to high stringency. However, such
hybridisation may be carried out under any suitable conditions known in the art (see
Sambrook et al, 1989. For example, if high stringency is required suitable conditions
include from 0.1 to 0.2 x SSC at 60°C up to 65°C. If lower stringency is required
suitable conditions include 2 x SSC at 60°C.
The coding ce of SEQ ID NO: 3 may be d by nucleotide
substitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100 substitutions. The
polynucleotide of SEQ ID NO: 3 may alternatively or additionally be modified by one
or more insertions and/or deletions and/or by an extension at either or both ends.
Additional sequences such as signal sequences may also be included. The modified
polynucleotide generally encodes a polypeptide which has endoglycosidase activity.
Alternatively, a polynucleotide encodes an epitope portion of an EndoS49
polypeptide. Degenerate substitutions may be made and/or substitutions may be made
which would result in a conservative amino acid substitution when the modified
sequence is translated, for e as shown in the Table above.
A nucleotide sequence which is capable of ively hybridizing to the
complement of the DNA coding sequence of SEQ ID NO: 3 will generally have at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at
least 99% sequence identity to the coding sequence of SEQ ID NO: 3 over a region of
at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably
at least 100 contiguous nucleotides or most preferably over the full length of SEQ ID
NO: 3. s for the calculation of sequence identity or similarity are sed in
more detail above in relation to the polypeptides of the invention.
Any combination of the above mentioned s of sequence identity and
minimum sizes may be used to define polynucleotides of the invention, with the more
stringent combinations (i.e. higher sequence identity over longer lengths) being
preferred. Thus, for example a polynucleotide which has at least 90% sequence
identity over 25, ably over 30 nucleotides forms one aspect of the ion, as
does a polynucleotide which has at least 95% sequence identity over 40 nucleotides.
Polynucleotide fragments, such as those suitable for use as probes or primers
will preferably be at least 10, preferably at least 15 or at least 20, for example at least
, at least 30 or at least 40 nucleotides in length. They will typically be up to 40, 50,
60, 70, 100 or 150 nucleotides in length. Probes and fragments can be longer than
150 tides in length, for example up to 200, 300, 400, 500, 600, 700 nucleotides
in length, or even up to a few nucleotides, such as five or ten tides, short of the
coding sequence of SEQ ID NO: 3.
Polynucleotides according to the invention may be produced inantly,
synthetically, or by any means available to those of skill in the art. They may also be
cloned by standard techniques. The polynucleotides are typically provided in isolated
and/or purified form.
In general, primers will be produced by synthetic means, involving a step wise
manufacture of the desired nucleic acid sequence one nucleotide at a time.
Techniques for accomplishing this using automated techniques are readily available in
the art.
Longer polynucleotides will generally be produced using recombinant means,
for example using PCR (polymerase chain reaction) cloning techniques. This will
involve making a pair of primers (e.g. of about 15-30 nucleotides) to a region of the
ndoS49 gene which it is desired to clone, bringing the s into contact with DNA
obtained from a bacterial cell, ming a polymerase chain reaction under
conditions which bring about amplification of the desired region, isolating the
amplified nt (e.g. by ing the reaction mixture on an agarose gel) and
recovering the amplified DNA. The primers may be ed to contain suitable
restriction enzyme recognition sites so that the amplified DNA can be cloned into a
suitable cloning vector.
Although in general the techniques mentioned herein are well known in the
art, reference may be made in particular to Sambrook et al, lar Cloning: A
Laboratory , 1989.
The polynucleotides according to the invention have utility in production of
the polypeptides according to the invention, which may take place in vitro.
cleotides ofthe ion may be used as a primer, e.g. a PCR primer, a primer
for an alternative amplification reaction, a probe e. g. labelled with a revealing label by
conventional means using radioactive or non-radioactive labels, or the
polynucleotides may be cloned into vectors.
cleotides or primers of the invention may carry a revealing label.
Suitable labels include sotopes such as 32P or 358, enzyme labels, or other
protein labels such as biotin. Such labels may be added to polynucleotides or primers
ofthe invention and may be detected using techniques known per se.
Polynucleotides or primers of the invention or fragments thereof, labelled or
unlabelled, may be used by a person skilled in the art in nucleic acid-based tests for
detecting or sequencing nd0S49 in a sample.
Such tests for detecting generally comprise bringing a sample containing DNA
or RNA into contact with a probe comprising a polynucleotide or primer of the
invention under hybridizing conditions and detecting any duplex formed between the
probe and nucleic acid in the sample. Such detection may be achieved using
techniques such as PCR or by immobilizing the probe on a solid support, removing
nucleic acid in the sample which is not ized to the probe, and then detecting
c acid which has hybridized to the probe. Alternatively, the sample nucleic acid
may be immobilized on a solid support, and the amount of probe bound to such a
support can be detected.
The probes of the invention may conveniently be packaged in the form of a
test kit in a le container. In such kits the probe may be bound to a solid support
where the assay formats for which the kit is designed requires such binding. The kit
may also contain suitable reagents for treating the sample to be probed, hybridizing
the probe to nucleic acid in the sample, control ts, instructions, and the like.
The polynucleotides of the invention may be incorporated into a recombinant
replicable vector. The vector may be used to replicate the nucleic acid in a compatible
host cell. ore, polynucleotides of the invention may be made by introducing a
polynucleotide of the invention into a replicable , introducing the vector into a
compatible host cell and growing the host cell under conditions which bring about
ation of the vector.
Preferably the vector is an expression vector sing a nucleic acid
sequence that encodes a polypeptide of the invention. Such sion vectors are
routinely constructed in the art of molecular biology and may for example involve the
use of d DNA and appropriate initiators, promoters, enhancers and other
elements, which may be necessary, and which are positioned in the correct
orientation, in order to allow for protein expression. Other suitable vectors would be
apparent to persons skilled in the art. By way of further example in this regard we
refer to Sambrook et al. 1989.
Polynucleotides according to the invention may also be inserted into the
s described above in an antisense orientation in order to provide for the
production of antisense RNA. nse RNA or other antisense polynucleotides or
interfering RNA, iRNA may also be produced by tic means. Such antisense
polynucleotides or iRNA may be used as test compounds in the assays ofthe
invention or may be useful in a method of treatment of the human or animal body by
therapy.
Preferably, a polynucleotide of the invention or for use in the invention in a
vector is operably linked to a control sequence which is capable of providing for the
expression of the coding sequence by the host cell, i.e. the vector is an expression
vector. The term “operably linked” refers to a juxtaposition wherein the components
described are in a relationship permitting them to on in their intended manner.
A regulatory ce, such as a promoter, “operably linked” to a coding sequence is
positioned in such a way that expression of the coding sequence is achieved under
conditions compatible with the regulatory sequence.
The vectors may be for example, plasmid, virus or phage vectors provided
with a origin of replication, ally a promoter for the sion of the said
polynucleotide and optionally a regulator of the promoter. The vectors may contain
one or more selectable marker genes, for example an llin resistence gene in the
case of a bacterial d or a resistance gene for a fungal vector.
Promoters and other expression regulation signals may be ed to be
compatible with the host cell for which expression is designed. For example, yeast
promoters include S. cerevisiae GAL4 and ADH promoters, S. pombe nmtl and adh
promoter. Mammalian promoters include the metallothionein promoter which can be
induced in response to heavy metals such as cadmium. Viral promoters such as the
SV40 large T antigen promoter or adenovirus ers may also be used. All these
promoters are readily available in the art.
Mammalian promoters, such as B-actin promoters, may be used. Tissue-
specific ers are especially preferred. Viral promoters may also be used, for
example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the
rous sarcoma virus (RSV) LTR promoter, the SV40 er, the human
cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV
IE promoters), or HPV promoters, particularly the HPV am regulatory region
(URR). Viral promoters are readily available in the art.
Expression s may be transformed into a suitable host cell to e for
sion of a polypeptide or polypeptide fragment of the invention. The host cell,
transformed or transfected with an expression vector as described above, is cultivated
under conditions to allow for expression of the polypeptide or fragment, and the
expressed polypeptide or fragment is recovered. Isolation and purification may be
carried out as described above. Host cells will be chosen to be compatible with the
vector and will preferably be bacterial. Host cells may also be cells of a non-human
animal, or a plant transformed with a polynucleotide of the invention.
IdiS
IdeS is an extracellular ne protease ed by the human pathogen S.
pyogenes and is described in WC 03/05 1914. IdeS was originally isolated from a
group A streptococcal strain of pe Ml, but the ides gene has now been
identified in all tested group A streptococcal strains. IdeS has an extraordinarily high
degree of substrate specificity, with its only identified substrate being IgG. IdeS
catalyses a single proteolytic cleavage in the lower hinge region of human IgG. This
proteolytic degradation promotes inhibition of opsonophagocytosis and interferes with
the killing of group A Streptococcus. IdeS also cleaves some subclasses of IgG in
various animals and efficiently converts IgG into Fe and Fab fragments. The ides
gene has been cloned and expressed in E. coli as a GST fusion n.
The IdeS polypeptide for use in the methods of the invention is preferably S.
pyogenes IdeS, or a variant or fragment of S. es IdeS which retains cysteine
protease activity. The t may be an IdeS ptide from another organism,
such as r bacterium. The bacterium is preferably a ococcus. The
Streptococcus is preferably a group A Streptococcus, a group C Streptococcus or a
group G ococcus. In particular, the variant may be an IdeS polypeptide from a
group C Streptococcus such as S. equiz' or S. zooepz'demz'cus. Alternatively, the variant
may be from Pseudomonas putida.
The IdeS polypeptide may comprise:
(a) the amino acid sequence of SEQ ID NO: 4;
(b) a variant thereof having at least 50% identity to the amino acid
sequence of SEQ ID NO: 4 and having IgG cysteine protease activity; or
(c) a fragment of either f having IgG cysteine protease activity.
Preferably, the IdeS polypeptide comprises, or consists of, the ce of
SEQ ID NO: 4. SEQ ID NO: 4 is the sequence of the mature form of IdeS, without
the signal sequence.
Variant IdeS polypeptides are those for which the amino acid sequence varies
from that in SEQ ID NO: 4, but which display the same IgG cysteine protease activity
as IdeS. Typically, polypeptides with more than about 50%, 55% or 65% identity,
preferably at least 70%, at least 80%, at least 90% and particularly preferably at least
95%, at least 97% or at least 99% identity, with the amino acid sequence of SEQ ID
NO: 4 are considered variants of the protein. Such variants may include allelic
variants and the deletion, modification or addition of single amino acids or groups of
amino acids within the protein sequence, as long as the peptide maintains the basic
functionality of IdeS. The ty of variants of SEQ ID NO: 4 may be ed
over a region of at least 50, at least 75, at least 100, at least 150, at least 200, at least
250, at least 275, at least 300 or more contiguous amino acids of the sequence shown
in SEQ ID NO: 4, or more preferably over the full length of SEQ ID NO: 4.
Variants of the amino acid sequence of SEQ ID NO: 4 preferably contain
residues Lys-55 and/or Cys-65 and/or His-233 and/or Asp-255 and/or Asp-257 of
SEQ ID NO: 4. Most preferably, the variant of SEQ ID NO: 4 contains each of
residues Lys-55, Cys-65, His-233, Asp-255 and 7 of SEQ ID NO: 4.
The variant sequences typically differ by at least 1, 2, 5, 10, 20, 30, 50 or more
mutations (which may be tutions, deletions or insertions of amino acids). For
example, from 1 to 50, 2 to 30, 3 to 20 or 5 to 10 amino acid substitutions, deletions
or insertions may be made. The modified polypeptide retains activity as an IgG-
specific cysteine protease. Preferably the variant polypeptides comprise a ne
residue and a histidine residue at a spacing typically found in ne ses. For
example, in SEQ ID NO: 4, these residues are found at a spacing of about 130 amino
acids, as is typically found in cysteine proteases.
The fragment of the IdeS polypeptide used in the invention is typically at least
, for example at least 15, 20, 25, 30, 40, 50 or more amino acids in , up to
100, 150, 200, 250 or 300 amino acids in length, as long as it retains the IgG cysteine
protease activity of IdeS. Preferably, the fragment of the IdeS polypeptide used in the
ion encompasses residues Lys-55 and/or Cys-65 and/or His-233 and/or Asp-255
and/or Asp-257 of SEQ ID NO: 4. Most preferably, the fragment encompasses each
dues Lys-55, Cys-65, His-233, Asp-255 and Asp-257 of SEQ ID NO: 4.
IdeS polypeptides for use in accordance with the invention display
immunoglobulin cysteine protease activity, and in particular IgG cysteine se
ty. Preferably, the polypeptide cleaves IgG in the hinge region and more
particularly in the hinge region of the heavy chain. Cleavage results in production of
Fe and Fab nts of IgG. Preferably the activity is specific for IgG. The cysteine
2012/067841
protease activity may be determined by means of a le assay. For example, a test
ptide may be incubated with IgG at a suitable temperature, such as 37°C. The
starting materials and the reaction products may then be analysed by SDS PAGE to
determine whether the desired IgG cleavage product is present. Typically this
cleavage product is a 3 lkDa fragment. Typically there is no further degradation of
IgG after this first cleavage. The cleavage product may be subjected to N—terminal
sequencing to verify that cleavage has occurred in the hinge region of IgG. Preferably
the N—terminal sequence comprises the sequence GPSVFLFP.
The cysteine protease activity ofthe polypeptides can be further characterised
by inhibition studies. Preferably, the activity is inhibited by the peptide derivate Z-
LVG-CHNZ and/or by iodoacetic acid both of which are protease inhibitors.
However, the activity is generally not inhibited by E64.
The ne protease activity ofthe polypeptides is generally ecific in
that the polypeptides may not degrade the other classes of Ig, namely IgM, IgA, IgD
and IgE, when ted with these immunoglobulins under conditions that permit
cleavage of IgG. The IdeS polypeptide is capable of ng human IgG. In
preferred embodiments the polypeptide has the ability to cleave human, rabbit, mouse
or goat IgG.
IdeS ptides for use in the present ion may be ed from any
suitable organism that expresses an IdeS polypeptide. Typically, the IdeS polypeptide
is isolated from suitable IdeS expressing strains of S. pyogenes. Suitable organisms
and strains may be identified by a number of techniques. For example, S. pyogenes
strains may initially be tested for the presence an ides gene. The presence of the ides
gene can then be verified by PCR using the primers or by hybridisation ofthe probes
to genomic DNA of the S. pyogenes strain.
S. pyogenes strains expressing active IdeS can be identified by assaying for
IgG cysteine protease activity in the culture supernatant. Preferably inhibitor E64 is
added to the supernatant to inhibit any SpeB cysteine se activity. At least five
s express active IdeS: strains APl, APl2, APSS, KTL3 and SF370. Preferably
the sing strain is selected from APl, AP12 and APSS.
Isolation and purification of IdeS from an expressing S. es culture, or
from cultures of other cells expressing IdeS is typically on the basis of IgG cysteine
protease activity. Preferably the purification method involves an ammonium sulphate
itation step and an ion exchange chromatography step. According to one
method, the culture medium is onated by adding sing amounts of
ammonium sulphate. The s of ammonium sulphate may be 10 to 80%.
Preferably the e medium is onated With 50% ammonium sulphate, and the
resulting atant is further precipitated With 70% ammonium sulphate. Pelleted
ptides may then be subjected to ion exchange chromatography, for e by
FPLC on a Mono Q column. Eluted fractions may be assayed for IgG cysteine
protease ty and peak activity fractions may be pooled. ons may be
analysed by SDS PAGE. For example, an N—terminal sequence can be obtained from
the SDS PAGE protein band. Fractions may be stored at -20°C.
Methods using the endoglycosidase activity 0fEnd0S49
As described herein, EndoS49 has endoglycosidase ty and is able to
hydroylse the glycan of glycoproteins including IgG and alpha-l-microglobulin. The
present invention thus provides methods for osylation of glycoproteins, and in
particular, hydrolysis of glycan from glycoproteins, and in particular, from IgG and
alpha-l-microglobulin. Typically, such a method includes incubating a sample
containing glycoprotein With EndoS49 With a glycoprotein under conditions Which
allow the endoglycosidase activity. Suitable conditions include use of EndoS49 at a
concentration of at least 1 ug/ml, 2 ug/ml, 4 ug/ml, 6 ug/ml, 8 ug/ml, 10 ug/ml, 12
ug/ml, 15 ug/ml or 20 ug/ml, preferably at least 10 ug/ml. Suitable conditions also
include incubation of the sample With EndoS49 for at least 20 minutes, 30 minutes, 40
minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes or 120 minutes,
preferably at least 60 minutes. Incubation preferably takes place at room temperature,
more preferably at approximately 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, and most
preferably at approximately 37°C.
These methods may be used to provide deglycosylated glycoproteins, Which
may themselves be useful in research or therapy. These methods may also be used to
characterise glycans on glycoproteins, for e, in glycomapping or
glycoprofiling. Such glycomapping and glycoprofiling is particularly useful for
antibody molecules, such as IgG molecules, for example, in the analysis of
monoclonal IgG molecules. Typically, the methods involved incubating the protein
With EndoS49 to hydroylase the glycans of the protein. Subsequently, the glycans and
2012/067841
the protein or polypeptide are separated, for example, using any suitable technique
such as HPLC or gel tography. The separated moieties can then be analysed
using any suitable analytical method, such as mass spectrometry, HPLC, gel
chromatography, gel electrophoresis, spectrometry, ary electrophoresis and other
standard laboratory techniques for the analysis of glycans and/or proteins.
In accordance with additional methods ofthe present invention, the methods
may also comprise utilising additional enzymes such as IdeS so that the glycans on
the Fc portion ofthe antibody can be analysed in more details using the methods and
ques described herein.
One example is to analyze the fucosylation of an immunoglobulin. The degree
of fucosylation on the Fc glycans on an IgG molecule is important for the therapeutic
potential of an IgG drug candidate. Afucosylated IgG molecules increase the ADCC
(nn) effect of the eutic IgG molecule. Thus, in accordance with the present
invention, there is provided a method for analyzing the amount of fucose in the Fc
glycans of an IgG, using EndoS49.
Typically, such a method includes incubating an glycoprotein, in this case an
immunoglobulin, with EndoS49 under conditions which allow the endoglycosidase
actiVity of 9. Suitable conditions include use of EndoS49 at a concentration
of at least 1 ug/ml, 2 ug/ml, 4 ug/ml, 6 ug/ml, 8 ug/ml, 10 ug/ml, 12 ug/ml, 15 ug/ml
or 20 ug/ml, preferably at least 10 ug/ml. Suitable ions also include incubation
ofthe sample with EndoS49 for at least 20 minutes, 30 minutes, 40 minutes, 50
minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes or 120 minutes, preferably
at least 60 minutes. Incubation preferably takes place at room temperature, more
preferably at approximately 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, and most
preferably at imately 37°C. IdeS may be added after the reaction with
EndoS49, or in the same reaction mixture, to induce proteolysis, ng the
immunoglobulin molecule into F(ab’)2 and Fe. The two nts are separated using
a separation method before ion into a mass spectrometer. After glycan cleavage,
a GlcNAc and core Fuc residue remain attached to Asn at the sus Fc/2
glycosylation site. Since an afocusylated immunoglobulin is not core fucosylated,
some Fc/2 will contain only a GlcNAc after digestion. The characteristic mass
difference (—146 Da) resulting from the absence of fucose is readily apparent in the
deconvoluted mass spectrum. Use of 9 therefore, facilitates the direct
estimation of the degree of core afucosylation of IgG.
Methodfor determining the presence or absence oflgG in a sample, orfor isolating
IgGfrom an IgG-containing sample
The isolation and/or detection of IgG is typically carried out in the art using
such agents as Protein G or n A. These bacterial proteins interact well with IgG.
However, Protein A does not bind to all four IgG subclasses (IgG1_4), and both Protein
A and Protein G are unable to minate between unglycosylated and/or denatured,
inactive IgG and glycosylated and/or , functionally active IgG. By contrast, the
present inventors have identified that EndoS49 polypeptides which lack IgG
endoglycosidase activity typically bind all four IgG subclasses with high affinity, and
are selective for normally glycosylated IgG, i.e. IgG in its native, onally active
form.
ingly, the present invention provides an ed method for
determining the ce or absence of IgG in a sample, which method comprises
contacting said sample with an EndoS49 polypeptide which lacks IgG
endoglycosidase activity, separating said EndoS49 from the contacted sample, and
thereby determining the presence or absence of IgG and, optionally, where IgG is
present, obtaining isolated IgG. The invention therefore also provides a method for
isolating IgG from an IgG-containing , which method comprises contacting
said sample with an EndoS49 polypeptide which lacks IgG endoglycosidase ty,
separating said EndoS49 from the contacted sample, and thereby obtaining isolated
IgG.
The above samples are contacted with EndoS49 polypeptide under conditions
suitable for interaction between the polypeptide and the sample to take place and IgG
binding activity to occur, i.e. to allow formation of a IgG-EndoS49 polypeptide
complex. Suitable conditions include use of EndoS49 at a concentration of at least 1
ug/ml, 2 ug/ml, 4 ug/ml, 6 ug/ml, 8 ug/ml, 10 ug/ml, 12 ug/ml, 15 ug/ml or 20 ug/ml,
preferably at least 10 ug/ml. Suitable conditions also include incubation of the
sample with EndoS49 for at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60
minutes, 70 minutes, 80 minutes, 90 minutes or 120 minutes, preferably at least 60
minutes. tion preferably takes place at room ature, more preferably at
approximately 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, and most preferably at
imately 37°C.
A particular age of EndoS49 in these methods is that EndoS49
specifically binds to normally glycosylated IgG. The IgG binding activities of other
IgG binding agents typically require or are enhanced by denaturation of the IgG
glycoprotein. This is lly achieved by treating an IgG-containing sample With
acid. Such treatment may damage or denature some antibodies sensitive
antibodies). Since the method of the invention requires no such treatment, the method
is particularly le for isolating acid-sensitive IgG in its native form from a
The EndoS49 may be separated from the contacted sample by any suitable
method. A red method for removal of the EndoS49 from a sample comprises
using an EndoS49 which is derivatised or modified as described above.
A preferred modification ses the addition of a histidine tag. The
presence of a histidine tag means that the polypeptide binds With a high y to a
reagent or separating means ning chelating groups on its surface Which carry a
nickel, copper or zinc ion. The histidine tag binds strongly to these metal ions. Such
a reagent can therefore be used to separate EndoS49 from a sample.
Another preferred modification comprises the addition of a biotin tag. The
presence of a biotin tag means that the polypeptide binds With a high y to a
reagent or separating means comprising streptavidin. The biotin tag binds strongly to
streptavidin. Such a reagent can therefore be used to separate 9 from a
sample.
Preferred reagents or separating means are populations of magnetic particles
capable of binding to the 9 polypeptide. For e, Where the EndoS49
polypeptide is derivatised With a histidine tag, the magnetic particles contain on their
surface chelating groups Which carry a nickel, copper or zinc ion. Alternatively,
Where the 9 polypeptide is derivatised With a biotin tag, the magnetic particles
contain on their surface streptaVidin.
Accordingly, a preferred method of removing EndoS49 from a sample
comprises using a population of magnetic particles as described above and carrying
out magnetic field separation on the sample. The magnetic particles are preferably
magnetic rticles, and the magnetic field separation is preferably high-gradient
magnetic field separation.
It will be understood that any suitable separation means may be used. For
example, the alternative means described in the preceding section.
The EndoS49 of the contacted sample may be assessed for the presence or
absence of bound IgG by any suitable means.
For example, the molecular weight of the EndoS49 may be ed.
EndoS49 bound to IgG will have a higher molecular weight than EndoS49 not bound
to IgG. Accordingly suitable methods include any method able to discriminate
protein species by , for example GE and Western Blot, Mass
spectrometry etc. Alternatively, the above n Blot may be directly analysed for
the presence of IgG by using IgG-specific antibodies or antibodies specific to a
ular IgG sub-class. ion ofproteins in a blot in this manner is a widely
used technique in the art.
Other suitable means for detecting the presence or absence of IgG bound to
EndoS49 include incubating the EndoS49 with antibodies to IgG or IgG-binding
proteins with coupled enzymes (e.g. horseradish peroxidase, alkaline phosphatase)
ed by addition of fluorogenic/chromogenic substrates. In this instance, the
development of a colour signal indicates the presence of IgG, with the quantity of IgG
being proportional to the strength of the signal. Detection of proteins in this manner
is a widely used que in the art.
Further suitable means for detecting the presence or absence of IgG bound to
EndoS49 comprise first ting bound IgG from EndoS49 so that it can be
analysed/detected independently of EndoS49 by any ofthe above methods or any
le method. IgG may be separated from EndoS49 by any suitable means.
Suitable means include the elution of IgG from EndoS49 by contacting the 9
from the contacted sample with a suitable elution buffer. The choice of elution buffer
will typically depend on whether or not the IgG bound to EndoS49 is known or
suspected to be acid-sensitive, i.e. denatured/inactivated by contact with acids.
Where the antibody is not acid-sensitive, an elution protocol using a low pH
elution buffer may typically be employed. Elution protocols of this type are well
known in the art. Such elution buffers have a pH typically below about pH 3, most
preferably below about pH 2. Preferred examples include 0.1 M Glycine at pH2. In
addition, or optionally, such elution buffers may typically comprise at least one of the
ing:
- Sodium or potassium salts, preferably at a concentration of about 0.5M to
about 1M;
- Mono-, di-, or polysaccharides with structures similar to the glycan
associated with Asn—297 on native IgG;
or any ation thereof.
However, as outlined above, the methods of the invention are ularly
suitable for detection/isolation of acid-sensitive antibodies. Where the IgG bound to
EndoS49 is known or suspected to be acid-sensitive, it is therefore preferable to use
n buffers and protocols which do not require a low pH. Such protocols are also
known in the art and are based on the principle ofproviding a buffer comprising a
le which competes with the bound IgG for binding to 9, thus leading to
release of the bound IgG. Suitable competition elution buffers therefore typically
comprise one or more mono-, di-, or polysaccharides with structures similar to the
glycan associated with 7 on native IgG. Particularly preferred elution buffers
comprise sucrose at about 0.25M to about 0.5M, preferably with pH from about 5.3 to
about 8.3. es of c red elution buffers include, for example:
Sucrose 0.25M, in PBS pH7.4; Sucrose 0.5M, in PBS pH7.4; Sucrose 0.25M, in PBS
pH5.3; Sucrose 0.25M, in PBS pH8.5 and Sucrose 0.25M, Maltose 0.25M, in PBS
pH7.4.
In addition, or optionally, such competition elution s may typically
comprise Sodium or potassium salts, preferably at a concentration of about 0.5M to
about lM.
The means for separating bound IgG from EndoS49 as described above may
also be used to obtain isolated IgG.
Method ofassessing the glycosylation state and/orfunctional quality ofan IgG
containing sample
The EndoS49 polypeptides of the invention in unmodified form have the
ability to hydrolyse glycan of IgG. Also, as described above, the EndoS49
polypeptides ofthe invention lacking IgG endoglycosidase activity and having IgG
binding activity are specific for glycosylated and/or , functionally active IgG.
Thus, the EndoS49 polypeptides can be used for analysing the glycosylation state of a
glycoprotein, and in particular an IgG antibody.
In accordance with one aspect of the ion, an IgG antibody is incubated
with an EndoS49 polypeptide of the invention which has endoglycosidase activity.
The products obtained can be analysed by any le techniques, including HPLC,
mass spec, gel chromatography, gel electrophoresis, spectrophotometer, capillary
electrophoresis. Such methods of analysis can be conducted at any stage in the
preparation of a protein, for example during screening of drug candidates, during
development of production processes ofbiologic drugs as well as a quality control in
release assays and during production.
The EndoS49 polypeptides which have been modified to remove or reduce the
endoglycosidase activity, or which retain the ability to bind to IgG in its native form
can also be useful in apping, and in ular the analysis of glycoprotein, and
in particular IgG structure.
For example, by using said 9 polypeptides, optionally in combination
with an alternative IgG-binding reagent, the present ion es a method of
assessing the glycosylation state or onal y of an IgG containing sample,
which method comprises taking a first and a second sub-sample from the IgG-
containing sample, contacting the first sub-sample with an EndoS49 polypeptide as
described in the preceding section and the second sub-sample with an alternative IgG-
binding reagent which is capable of binding unglycosylated and/or denatured, inactive
IgG, and then quantifying the amount of IgG bound to the EndoS49 polypeptide in the
first sub-sample, and the amount of IgG bound to the alternative IgG-binding reagent
in the second sub-sample. Finally, by comparing both of the amounts of bound IgG
determined in the first and second sub-samples, the glycosylation state or functional
quality of an IgG containing sample can be assessed.
The alternative IgG-binding reagent is typically Protein A or Protein G, which
bind to all forms (native or denatured) of IgG. In this instance, the amount of IgG
bound to said reagent therefore represents the total IgG present in the second sub-
sample. The EndoS49 polypeptide binds only to glycosylated and/or native,
onally active IgG, and therefore the amount of IgG bound to EndoS49
represents only the glycosylated and/or native, functionally active IgG present in the
first sub-sample. By comparing the concentration of total IgG from the second sub-
sample to the concentration of native IgG in the first sample, the skilled person will
ise that one obtains a ratio which reflects the proportion of IgG in the al
sample which is present in its glycosylated and/or native, onally active form.
In another embodiment, the alternative nding reagent could be specific
for unglycosylated and/or denatured IgG. Such a reagent could be, for example, an
antibody. Accordingly, in this embodiment the proportion of IgG in the al
sample which is present in its glycosylated and/or native, fi1nctionally active form can
be assessed by the formula:
Amount ofIgG infirst sub-sample/(Amount ofIgG infirst sub-sample + Amount of
IgG in second sub-sample)
The samples in the above methods are contacted With EndoS49 polypeptide or
alternative nding reagent under conditions le for interaction between the
polypeptide or reagent and the sample to take place and IgG binding ty to occur.
Suitable conditions are, for example, equivalent to those set out in the preceding
section.
Methodfor isolating IgG Fab or F6fragmentsfrom an IgG-containing sample
The methods of the present invention can be used for isolating Fab fragments
from IgG-containing s. In one embodiment, the present invention provides a
method for isolating Fab fragments of IgG from an IgG-containing sample, Which
method comprises:
(a) contacting said IgG-containing sample With IdeS, and an EndoS49
ptide;
(b) separating said IdeS and said EndoS49 ptide from the contacted
sample; and
thereby ing Fab fragments.
Preferred methods for ting IdeS and EndoS49 polypeptide from a
sample comprises using an IdeS and/or EndoS49 polypeptide Which is derivatised or
modified as described above. The same or a different ation may be used on
each of IdeS and the EndoS49 polypeptide.
A preferred modification comprises the addition of a histidine tag. The
presence of a histidine tag means that the polypeptide binds With a high affinity to a
reagent or separating means containing chelating groups on its surface Which carry a
nickel, copper or zinc ion. The histidine tag binds strongly to these metal ions. Such
a reagent can therefore be used to separate IdeS and/or EndoS49 polypeptide from a
sample.
Another preferred modification comprises the addition of a biotin tag. The
presence of a biotin tag means that the polypeptide binds With a high affinity to a
t or separating means sing streptavidin. The biotin tag binds strongly to
streptavidin. Such a reagent can therefore be used to separate IdeS and/or EndoS49
polypeptide from a sample.
Preferred reagents or separating means are populations of magnetic particles
capable of binding to the EndoS49 polypeptide. For example, where the IdeS and/or
EndoS49 polypeptide polypeptide is derivatised with a histidine tag, the magnetic
particles contain on their surface ing groups which carry a nickel, copper or zinc
ion. Alternatively, where the IdeS and/or EndoS49 polypeptide polypeptide is
derivatised with a biotin tag, the magnetic particles contain on their surface
streptavidin.
ingly, a preferred method of ng EndoS49 from a sample
comprises using a population of magnetic particles as bed above and carrying
out magnetic field separation on the . The magnetic les are preferably
magnetic nanoparticles, and the magnetic field separation is preferably high-gradient
magnetic field separation.
Thus, step (a) of the above method preferably additionally comprises
contacting the sample with a population of magnetic nanoparticles capable of binding
to IdeS and the EndoS49 polypeptide, and n step (b) comprises carrying out
magnetic field tion on the sample.
The EndoS49 polypeptide is preferably a modified EndoS49 polypeptide
lacking endoglycosidase activity.
In the above ment of the invention, the ntaining sample
typically ses purified or ed IgG. By "purified or ed IgG" is meant
an IgG fraction with a purity of normal commercial grade. IgG is typically isolated
from a sample such as serum or, in the case of recombinant IgG, from cell lysate.
Isolation may be carried out according to any suitable method, preferably according to
the method described above for the isolation of IgG using a modified EndoS49
polypeptide lacking endoglycosidase activity. Thus, one embodiment of the invention
encompasses the method set out above comprising, prior to step (a):
(i) contacting said IgG-containing sample with an EndoS49 polypeptide
which lacks IgG endoglycosidase activity, to thereby allow formation of a
IgG-EndoS49 polypeptide complex;
(ii) separating said doS49 polypeptide complex from the contacted
sample;
(iii) eluting IgG from the IgG-EndoS49 polypeptide complex thereby
obtaining an IgG-containing sample;
and wherein steps (a) and (b) are carried out the IgG-containing sample obtained in
step (iii). Separation ofthe doS49 polypeptide complex from a sample is
preferably carried out according to the methods set out in the section above relating to
methods for determining the ce or absence of IgG in a sample, or for isolating
IgG from an IgG-containing sample.
In an alternative embodiment of the invention, the methods are adapted to
isolate Fab fragments from IgG-contained samples t the need to purify the IgG
before carrying out the method. These methods can be carried out on a sample
ning unpurified IgG, for e, whole serum, cell lysate or cell culture
medium. In this embodiment of the invention, the method comprises:
(a) contacting said IgG-containing sample with an EndoS49 polypeptide to
thereby allow formation of a IgG-EndoS49 polypeptide complex;
(b) separating said IgG-EndoS49 polypeptide complex from the contacted
sample;
(c) adding to doS49 ptide complexes obtained in step (b)
IdeS; and (d) separating said IdeS and said EndoS49 polypeptide from the
mixture obtained in (c);
and thereby isolating Fab fragments.
The methods for separating IdeS and/or EndoS49 polypeptide from the
samples/mixtures above preferably comprise using an IdeS and/or 9
polypeptide which is derivatised or modified as described above. The same or a
different modification may be used on each of IdeS and the EndoS49 polypeptide.
Preferred reagents or separating means are tions of magnetic particles capable
ofbinding to the IdeS and/or 9 polypeptide. For example, where the IdeS
and/or EndoS49 polypeptide polypeptide is derivatised with a histidine tag, the
magnetic particles contain on their e chelating groups which carry a nickel,
copper or zinc ion. Alternatively, where the IdeS and/or EndoS49 polypeptide
polypeptide is derivatised with a biotin tag, the magnetic particles contain on their
surface streptavidin.
Thus, step (a) of the above method preferably additionally comprises
contacting the sample with a population of magnetic nanoparticles capable of binding
to the EndoS49 polypeptide, step (c) additionally ses contacting the IgG-
EndoS49 polypeptide complexes obtained in step (b) with a population of magnetic
nanoparticles e of binding to IdeS and the EndoS49 polypeptide, and wherein
steps (b) and (d) comprise carrying out magnetic field separation on the sample of (a)
and mixture obtained in (c), respectively.
It will be understood that any suitable separation means may be used. For
example, the alternative means described in the section relating to methods for
ing a population of cells which are substantially free of IgG molecules bound to
FcyRs could be adapted for separation of IdeS and/or EndoS49 polypeptide.
The EndoS49 polypeptide in the above embodiment is preferably a modified
EndoS49 polypeptide g endoglycosidase activity.
The above methods of the invention can also be used for isolating Fc
fragments from IgG-containing samples. In one such embodiment of the invention,
the method comprises:
(a) ting said IgG-containing sample with IdeS;
(b) separating IdeS from the mixture obtained in step (a), thereby isolating
Fab and Fe fragments;
(c) ting said Fab and Fe fragments with an 9 polypeptide to
thereby allow ion of a PC fragment-EndoS49 polypeptide complex;
(d) separating the Fc fragment-EndoS49 polypeptide complexes from the
mixture obtained in step (c); and
(e) ing Fc fragments from the Fc fragment-EndoS49 polypeptide
complexes obtained in step (d).
It will be understood that any suitable separation means may be used as
described above, however, the methods for separating IdeS and/or EndoS49
polypeptide from the samples/mixtures above preferably comprise using an IdeS
and/or EndoS49 ptide which is derivatised or modified as bed above.
Preferably, step (a) of the above method additionally comprises contacting the
sample with a population of magnetic nanoparticles e of binding to IdeS, step
(c) onally comprises contacting the Fab and Fe fragments obtained in step (b)
with a tion of magnetic nanoparticles capable of binding to the EndoS49
polypeptide, and wherein steps (b) and (d) comprise carrying out magnetic field
separation on the sample of (a) and mixture obtained in (c), respectively. The
EndoS49 polypeptide is preferably a modified EndoS49 polypeptide lacking
endoglycosidase activity.
In an alternative ment, Fc fragments may be isolated from an IgG-
containing sample by a method comprising:
(a) contacting said IgG-containing sample with IdeS and an EndoS49
polypeptide
(b) ting the EndoS49 polypeptide from the e obtained in (a);
thereby isolating Fc fragments.
It will be tood that any suitable separation means may be used as
described above. However, preferably step (a) of the above method onally
comprises contacting the sample with a population of magnetic nanoparticles capable
ofbinding to the EndoS49 polypeptide but not to IdeS, and wherein step (b)
comprises ng out magnetic field separation on the mixture obtained in (a).
Preferably, the IdeS and/or the EndoS49 polypeptide are derivatised or modified as
described above, with the proviso that a different modification is applied to each. For
example, where the IdeS is modified by addition of a ine tag such that it binds to
a population of magnetic particles containing on their surface chelating groups which
carry a nickel, copper or zinc ion, the EndoS49 polypeptide might be modified by
addition of a biotin tag such that it binds to a population of ic particles
containing on their surface streptavidin. The EndoS49 ptide is preferably a
modified EndoS49 polypeptide lacking endoglycosidase activity.
Similar to the methods for isolating Fab nts, it will be iated that
in the methods for separating Fc fragments the IgG-containing sample typically
comprises purified or isolated IgG. A preferred method of isolating IgG is described
in steps (i) to (iii) as set out above.
The present invention es
- a kit for isolating IgG from an IgG-containing sample, comprising:
(a) an EndoS49 polypeptide according to the ion which lacks
endoglycosidase activity; and optionally
(b) means for separating said EndoS49 polypeptide from a sample.
- a kit for determining the presence or absence of IgG in a sample, comprising:
(a) an EndoS49 polypeptide according to the invention which lacks
endoglycosidase activity; and optionally
(b) means for separating said EndoS49 polypeptide from a sample.
- a kit for assessing the glycosylation state and/or functional quality of an IgG
containing sample, comprising:
(a) an EndoS49 polypeptide according to the ion which lacks
endoglycosidase activity; and optionally;
(b) an ative IgG-binding reagent which is capable of g
denatured and/or deglycosylated IgG;
(c) means for separating said 9 polypeptide from a sample; and
(d) means for separating said alternative IgG-binding reagent from a
sample.
The ative IgG-binding reagent comprises Protein G and/or Protein A
and/or Protein A/G.
The present invention also provides:
- a kit for isolating Fab or Fc fragments of IgG comprising:
(a) IdeS;
(b) an EndoS49 polypeptide; and
(c) means for ting said IdeS and said EndoS49 polypeptide from a
sample.
In a preferred ment, the kit additionally comprises an 9
polypeptide according to the invention which lacks endoglycosidase activity and a
means for separating said EndoS49 ptide from a sample. The 9
polypeptide is preferably an EndoS49 polypeptide according to the invention which
lacks endoglycosidase activity.
Preferred embodiments of the above kits further comprise instructions for
using the kit in a method of the invention. Further preferred embodiments include
those wherein the means for separating an EndoS49 polypeptide, an alternative IgG-
binding reagent, an IdeS polypeptide, or an EndoS49 polypeptide from a sample are
populations of magnetic nanoparticles, wherein each population is capable ofbinding
to at least one of the ted polypeptides/reagents/proteins. In this embodiment the
kit typically additionally comprises instructions to perform magnetic field separation
on the sample.
In preferred embodiments of the above methods and kits, the
polypeptides/proteins/reagents used are derivatised with an y tag, preferably a
histidine tag, to assist with separation of said polypeptides.
The following Examples illustrate the invention:
Example 1
MATERIALS AND METHODS
Bacterial strains and growth
The genome ofGAS strain NZl3l of serotype M49 has been sequenced and
this strain was therefore selected as reference strain in this work (McShan et (11., 2008)
(Chaussee et al., 1999). GAS strain NZl3l was propagated on blood agar
andEscherz'chz'a coli strains ToplO (Invitrogen) and BL2l pLysS (Invitrogen) were
propagated on lysogeny broth (LB) agar. For selection in E. coli ToplO cells,
carbenicillin was used at 100 ug/mL and for E. coli BL2l pLysS, 100 ug/mL
carbenicillin and 34 ug/mL chloramphenicol were used. ght cultures of E. coli
were carried out in LB at 37°C with aeration. Genomic DNA preparation of GAS
strain NZl3l was med using Puregene DNA Purification Kit (Qiagen).
Transformation was carried out using heat-shock at 42°C for 30 s. Plasmid
preparations from E. coli were med using Plasmid Miniprep Kit I (E.Z.N.A).All
primers used in this work are listed in Table 2.
Recombinant expression of EndoS49
Recombinant expression of EndoS49 in E. coli was established by PCR
amplification of the nd0S49 gene from group A Streptococcus strain NZl3 l , serotype
M49 with the primers ndoS49-F-BamHI, CTGTAAGGATCCAGGAGAAGACTG,
and -R-Xhol, GAAACCTCGAGTCTTTGTAATCGTAGGACTT. The
nd0S49 gene nt was digested with restriction enzymes BamHI and XhoI and
ligated into the sion vector pGEX-SX-3 (Amersham Biosciences) using DNA
ligase T4 (Fermentas) creating the d pGEX-ndoS49. The sion vector was
transformed into E. coli ToplO chemically competent cells and recombinant cells
were grown on 100 ug/mL carbenicillin plates and screened with PCR using primers
ndoS49-F-BamHI and ndoS49-R—XhoI. Positive clones were isolated and the pGEX-
ndoS49 plasmid was purified and transformed into the E. coli expression strain BL2l
pLysS as described above.
One recombinant clone was grown overnight at 37°C with antibiotics and
diluted 1:20 in LB medium with antibiotics and grown for 3 h. The expression of the
n EndoS49 was induced with 0.1 mM IPTG for 3 h. The cells were harvested
and lysed with BugBuster Protein tion Reagent (Novagen). Recombinant GST-
EndoS49 was purified on column with Glutathione Sepharose 4B (GE Healthcare)
and d with reduced glutathione.
Mutagenesis of EndoS49
Site-directed mutagenesis of the glutamic acid 186(Glu-l86) to leucine
(El86L) was carried out using QuickChange II Site-Directed Mutagenesis Kit
(Stratagene) ing to the manufacturer’s ctions. The mutagenesis primer
used was CTAGATATTGATATTmCACGAATTTACGAAC in combination with
the antisense of the ce above and the plasmid pGEX-nd0S49(mutation
underlined). This generated the plasmids d0S49(El 86L) and, after
sequencing, recombinant EndoS49(El86L) was expressed and d as described
for EndoS49.The truncated versions of EndoS49 were constructed by amplifying parts
ofthe nd0S49 gene from GAS NZl3l with primersndoS49(truncl-5) containing
restrictions sites BamHI and XhoI (Table 2). The fragments were digested and ligated
into the pGEX vector as above, and transformed into E. coli ToplO and subsequently
to BL21 pLysS and grown with antibiotics. The proteins were produced as above and
the proteins EndoS49(trunc1) 80 kDa, EndoS49(trunc2) 70 kDa, EndoS49(trunc3) 60
kDa, EndoS49(trunc4) 50 kDa, EndoS49(trunc5) 42 kDa were purified.
Glycoprotein glycan hydrolysis assay
1 ug recombinant EndoS49and its mutants were incubated with 3 ug of each
glycoprotein in 20 uL PBS overnight at 37°C. Glycan ysis was analyzed on a
% SDS-PAGE gel and subsequently analyzed with LCAlectin blot as previously
described (Collin and Olsén, 200 la).
Slot-blot analysis
EndoS49 and its mutants were immobilized on a methanol activated PVDF
membrane at 4, 2, 1 ug in PBS per slot using Millipore slot blot equipment. The
membrane was blocked with 5% skim milk (Difco) for l h at room temperature.
Washing was consistently carried out for 3x10 s in PBST. The membrane was
incubated with 10 ug human IgG (Sigma) in 0,5% skim milk for l h at 37°C and then
washed. 5 ug horseradish peroxidase conjugated with protein G rogen) was
added to the membrane and incubated for l h at 37°C. After washing the membrane
was developed with Supersignal West Pico Chemiluminiscent ate o
Scientific).
Bioinformatic analysis
The genes ndoS49 and ndoS were translated into EndoS49 and EndoS and
compared using the ClustalW algorithmin within the software MacVector (MacVector
Inc.). The phylogenetic tree was constructed with MacVector using protein ces
from NCBI PubMed with the following accession numbers: EndoS (AF296340),
EndoE (AAR20477), EndoH (NP_631673), EndoC (ADC53484), EndoF2 (P369l2),
EndoF3 (P36913) n and Olsen, 2001b) (Collin and Fischetti, 2004) (Tarentino
and r, 1974) (Tarentino et al., 1993).
PCR screening for ndoS49
s amplifying the ndoS49 gene from GAS were designed and denoted
ndoS49-F (AAAACGCGGACCACTATATGC) and ndoS49-R
(AAACGTTGTCCGAGGATTTG). 42 GAS strains were propagated on blood agar
and grown overnight at 37°C with 5% C02. Single colonies were picked and lysed in
uL sterile H20 at 99°C for 10 minutes. These lysates were used as template for a
stringent PCR reaction to detect ndoS49 in the 42 GAS strains. As a positive control,
primers for the amplification of the gene recA were designed, recA-F
(AGCCCTTGATGATGCTTTG) and recA-R (AACAATTCTGGGTGATCGG).As
positive controls, both PCR reactions used genomic DNA from GAS strain NZ131
(M49) and APl (M1) as template.
S
ClustalW analysis reveals two different enzymes: EndoS49 and EndoS
The genes ndoS49 and ndoS were in silico translated into proteins and
ed using the ClustalWalgorithm. On the gene level the identity is 50% and
37% on the protein level. The ClustalW analysis revealed a (nearly) identical signal
peptide sequence and a conserved family 18 glycoside hydrolasecatalytic domain
(DGLDIDIE)(Figure 1). Experimental analysis of EndoS has shown that tryptophans
are essential for the glycan-hydrolyzing activity (Allhom et al., 2008). These
tryptophans are also conserved in EndoS49.
WO 37824
Recombinant EndoS49 show glycan yzing activity on human glycoproteins
The 90 kDa 9 was successfully recombinantly expressed in E. coli
BL21 and purified from the soluble fraction using the GST-tag. EndoS49(El86L), a
catalytic mutant with the glutamic acid of the GHl 8 motif (El 86) substituted for a
leucine (L), was constructed and purified in the same way. To map the activity of the
protein, 5 carboxy-terminally truncated versions of the s were constructed and
denoted EndoS49(truncl) 80 kDa, EndoS49(trunc2) 70 kDa, 9(trunc3) 60
kDa, EndoS49(trunc4) 50 kDa and EndoS49(trunc5) 42 kDa. This collection of
enzymes was utilized to analyze the glycan hydrolyzing activity of EndoS49 on
human glycoproteins. First, the enzymes were incubated with human IgG overnight
and analyzed on a SDS-PAGE gel and with LCA lectin blot, detecting the mannose
structures in the glycan of IgG. The gel revealed a shift of 4 kDa of the IgG heavy
chain treated with EndoS49 and the LCA lectin blot confirmed this shift as a lack of
the ed glycan (Figure 2A). EndoS49(El 86L) showed no shift and no change in
glycan composition suggesting that El 86 plays a crucial role in the catalytic activity
of EndoS49. Concerning the truncated enzymes, EndoS49(truncl-4) showed activity
on the glycan of IgG but EndoS49(trunc5), the smallest of the enzymes (42 kDa),
showed no glycan-hydrolyzing activity e 2A).
Further analysis of the IgG deglycosylation by EndoS49 was carried out by
incubating IgG1_4 with EndoS49 and EndoS49(El86L), ght. The IgG subclasses
were analyzed as above and showed that EndoS49 has activity on all four subclasses
ofIgG, and in line with previous , the catalytic mutant showed no activity
e 2B). Incubating the collection of enzymes with alpha-l-microglobulin, a
heavy glycosylated human serum protein, and analysis on SDS-PAGE showed glycan
hydrolysis activity of EndoS49on this glycoprotein(Figure 2C). To further elucidate
the specificity of EndoS49, a model substrate consisting of a N-acetyl—beta-D-
glucosaminide d to the fluorescent 4-methylumbelliferylwas incubated with
EndoS49 for l, 2, 3, 4 and 16 h and the fluorescence measured. The fluorescence will
se if the sugar is cleaved but no such increase in intensity was observed (data
not shown) suggesting that 9 has activity only on glycoprotein ates.
EndoS49 binding to IgG
The finding that EndoS49 has glycan hydrolyzing activity on IgG led us to
believe that the enzyme binds IgG. This was evaluated with slot blot analysis where
EndoS49 and its catalytic mutant and ted versions were immobilized on a
PVDF ne and incubated with IgG and the binding detected with protein G
coupled to Horseradish-peroxidase (HRP). The slot blot show an increased binding to
IgG by the tic mutant EndoS49(El86L) (Figure 3).
The gene ndoS49 is present in GAS serotype M49
To elucidate whether ndoS49 is t in any other serotypes than M49 a
stringent PCR was deployed to analyze the presence of the ndoS49 gene in a ion
ofGAS strains. The primers ndoS49-F and ndoS49-R was used in a PCR on lysates
from GAS colonies together with the positive control amplifying the recA gene,
present in all GAS strains. The ndoS49 gene was amplified in all selected GAS M49
serotypes and also in serotype M60, whereas no other serotype gave a PCR product
(Table 1).
In the sequenced genomes of GAS strains NZ131 (M49) and MGASSOOS
(M1) the genes surrounding ndoS49 and ndoS were compared revealing that the genes
are located in the same genomic context and that the surrounding genes are highly
conserved (Figure 4). The full length EndoS49 was compared to a selection of
usly described endoglycosidases, EndoS, EndoC, EndoH, EndoE, EndoF2,
EndoF3) and a phylogenetic tree was reconstructed (Figure 5). This revealed that
EndoS and EndoC are more y related than EndoS and EndoS49 and that
endoglycosidases from Streptococcus are close related compared to enzymes from
other bacteria.
Table 1. The presence of ndoS49in a selection of GAS serotypes
Strain Serotype ndoS49 recA
5448 V11 --
SF370 V11 --
ACN1 V11 --
ACN2 V12 --
ANC3 V13 --
20224 V13 --
ACN4 V14 --
ACNS V15 --
Manfredo V15 --
ACN6 V16 --
AP6 V16 --
ACN9 V19 --
ACN1 1 V11 1 --
ACN12 V112
ACN18 V118
ACN19 V119
ACN22 V122
ACN24 V124
ACN28 V128
NZ131 V149
3487-05 V149
AWl V149 -- --
AW2 V149 -- --
AW3 V149 -- --
AW4 V149 -- --
AW6 V149 -- --
AW7 V149 -- --
AW8 V149 -- --
AW9 V149 -- --
AWl0 V149 -- --
AWl 1 V149 -- --
AW12 V149 -- --
AW13 V149 -- --
ACN49 V149 -- --
AP49 V149 -- --
CS 1 0 1 V149 -- --
AP53 V153 --
ALAB49 V153
ACN55 V155
ACN57 V157
ACN60 V160
AP74 V174
Table 2. Primers used in this work
Primer name Sequence (5’-3’)
ndoS49-F- CTGTAAGGATCCAGGAGAAGACTG
BamHI
-R-Xh01 GAAACCTCGAGTCTTTGTAATCGTAGGACTT
ndoS49(E1 86L)- CTAGATATTGATATTCTTCACGAATTTACGAAC
ndoS49(E186L)- GTTCGTAAATTCGTGAAGAATATCAATATCTAG
ndoS49(trunc 1 )- ATTTCTCGAGCTGAAGACGTCCTTTAGCCACG
R-Xhol
ndoS49(trunc2)- TAAACTCGAGCCCCATCAGAAACATCTACTAAG
R-Xhol
(trunc3)- ATTTTCTCGAGGCATTATCAACATCATAATGACC
R-Xhol
ndoS49(trunc4)- TAAACTCGAGCCAGTCATGCCTACCATAACAAGCTCAGC
R-Xhol
ndoS49(truncS)- ATTTCTCGAGCTGTCCAACTTGTTGAATG
R-Xhol
ndoS49-F AAAACGCGGACCACTATATGC
ndoS49-R AAACGTTGTCCGAGGATTTG
recA-F AGCCCTTGATGATGCTTTG
recA-R AACAATTCTGGGTGATCGG
The protein sequence of EndoS49
NCBIReferenceSequences
NZl3l genome: NC_011375.1
ndoS49 gene sequence: NC_01 1375.1
9 protein sequence: YP_0022863 83.1
EndoS49 Protein Sequence:
MDKT. .VKRT TLWGAAJAT{{DSLNTVKAT.KTVQTG
KTDQQVGAKT.VQ«. PLYAGYFRTWTDRASTGIZDGKQQ{PTVTWATVPKTVD
LFVFTDHTASDSPFWSTLKDSYVTKgiQQGTA'.VQT GVVT.NGRTG;SK3YPDTPTG
NKAUAAA"VKAFVTDRGVDGLD 3 TitTVKTTP««3ATAHNVtKT AQH GKVGSD
KSKHH"M3TTHSV«.VVP tKG ATDUDY..RQYYGSQGGTATVDT VSDWVQYQVYID
ASQtqutSttTTSASKGV;WFDVVTYDPVVP«.KGKD TGTRAKKYATWQPSTGGgKA
G hSYA DRDGVATVPSTYKNRTSTVHQRTTVDN STTDYTVSRK.KTHMTTDKRYDV
DQKD PDPAURTQ KGDHTRYVKT VTTGDK"QV.KG.TKUSKHQKUTUR
Q .SNVKT TPTUUPTSMKKDATUVWVGWTG TKT.VT.SG.VRQTUDG DVNS THUTSF
D'SHVS.DHSTKSTDRKLLWTUMTQVSV{QKITVKNTAFTVQKPKGYYPQTYDTKTGT
YDVDVATTD UTDtVtGTVTKRNTt GDTTAtA YKTGAVDGRQYVSKDYTYTAFRKD
YKGYKV{HTASNUGTTVTSKVTATTDTTYUVDVSDGTKVVTTWKUN GSGA WMTVLA
KGAKV: GTSGDtTQAKK hDG.KSDthTWGQTNW AtDHGT NHAKTWTHtVATTNT
«. KTDSSJVVAKGRHQ'TKDTT DU«.KWD KVRKTYUSNDTVWTDVAQWDDAKAIFNS
KLSNVLSTYWTFCVDGGASSYYPQYTTLQ LGQRHSVDVAVTLK)
Example 2
Monoclonal lg les can show minor variations in the Fc glycans and as a
consequence the Fc s can appear as an inhomogeneous pool of Fe glycans. The
vast majority ofthe glycans are identical but a minority can show variable
carbohydrate structure or composition. The variety arises both from the origin of the
Fc part, which can be human, humanized or from another species, and from the choice
uction ine and cell culture ions. In this example two well-known
IgG based drugs, Avastin and Erbitux, were deglycosylated both with EndoS and with
EndoS49. The samples were incubated with EndoS or EndoS49 as set out in the
Table below.
2012/067841
Lane Sample Erbitux Avastin EndoS EndoS49 ideS
100 pg 100 pg 0.1 mg/ml 0.1 mg/ml 1 pg
(H1) (H1)
1 MW standard - - - - - --
3 -- --
4 -- --
- - 5
-- --
- - 50
6 __ __
_ _ _
7 -- --
8 -- --
- 50
9 -- --
- - 5
l 0 -- --
- - 5 0
l l -- --
- - -
12 MW standard - - - - -
The s are presented in Figure 6. It was found that EndoS49 has the
potential to cleave a larger variety of PC glycans than EndoS. Even if incubation was
left over night in order to minimize the effect of potential differences in the enzymatic
activities the EndoS enzyme could not fully deglycosylate Erbtux Fc glycans.
However EndoS49 shows a complete deglycosylation profile and is hence the more
favourable enzyme when it comes to glycan profiling of immunoglobulins.
Claims (9)
1. An isolated polypeptide comprising (a) the amino acid sequence of SEQ ID NO: 1; (b) a variant thereof having at least 95% identity to the amino acid sequence of SEQ ID NO: 1 over at least 810 contiguous amino acids of SEQ ID NO: 1 and having the endoglycosidase activity of a polypeptide ting of the amino acid sequence of SEQ ID NO: 1.
2. A polypeptide according to claim 1 which consists of the amino acid sequence of SEQ ID NO: 1.
3. An isolated polypeptide e of binding to IgG and which does not have endoglycosidase activity comprising (a) the amino acid sequence of SEQ ID NO: 2; (b) a variant thereof having at least 95% identity to the amino acid sequence of SEQ ID NO: 1 over at least 810 contiguous amino acids of SEQ ID NO: 1, in which the amino acid lent to ic acid at position 186 is substituted.
4. A method for hydrolysing glycan of a glycoprotein comprising incubating the glycoprotein with a polypeptide as defined in claim 1 or 2.
5. A method according to claim 4, wherein the glycoprotein is completely deglycosylated.
6. A method for assessing the glycosylation status of a glycoprotein comprising ting a glycoprotein with a polypeptide according to claim 1 or 2, and analysing the products ed.
7. The method according to claim 6 which comprises: (a) ting the glycoprotein with the said polypeptide to hydrolyse glycan of the glycoprotein; (b) separating the glycan from the deglycosylated protein; (c) analysing the glycan and/or deglycosylated protein so produced.
8. The method according to any one of claims 4 to 7 wherein the glycoprotein comprises an IgG antibody or a monoclonal IgG antibody.
9. An isolated polypeptide according to any one of claims 1 to 3 substantially as herein described and with or without nce to any one or more of the es and/or
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB1115841.7 | 2011-09-13 | ||
GB201115841A GB201115841D0 (en) | 2011-09-13 | 2011-09-13 | Protein and method |
PCT/EP2012/067841 WO2013037824A1 (en) | 2011-09-13 | 2012-09-12 | Endoglycosidase from streptococcus pyogenes and methods using it |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ622172A NZ622172A (en) | 2016-04-29 |
NZ622172B2 true NZ622172B2 (en) | 2016-08-02 |
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ID=
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