CA2064818A1 - Fibulin - Google Patents

Abstract

The invention is directed to a substantially purified protein, hereinafter termed ''Fibulin'', that interacts with the cytoplasmic domain of the .beta. subunits shared by the fibronectin receptor and some other integrins. This invention is further directed to isolates of nucleic acid encoding Fibulin and to expression vectors harboring such nucleic acid in expressible form, to microorganism strains or cell cultures transformed with them and to recombinantly produced Fibulin obtainable via expression of DNA encoding Fibulin in a transfected recombinant host system. Still further the invention is directed towards antibodies reactive with Fibulin, be they monoclonal or polyclonal, and to nucleotide sequences capable of specifically hybridizing with nucleic acids encoding Fibulin.

Description

WO 91/027~ 2 0 ~ 4 8 1 8 PCT/US90/04662 FIBULIN

BACXGROUND OF THE INVENTION

This invention relates generally to cell adhesion systems, and more specifically, to a protein which interacts with the cytoplasmic domain of certain adhesion receptors.

Multicellular organisms, such as man, have some 10l4 cells whlch may be divided into a minimum of fifty types, such as blood cells and nerve cells, etc. During the course of growth and development, cells adhere to other cells, or to extra-cellular materials, in specific and orderly ways. Although the interactions of cells with one another and with extracellular matrices are not well understood, they play an important role in the life of the cell. The adhesion of cells to other cells or Pxtracellular substrates appears to be mediated by specific cell surface receptors which bind to specific ligands.

The fibronectin receptor is a heterodimer of two transmembranous subunits, ~ and B. Of these, the ~ subunit is unique to the fibronectin receptor while the ~ subunit, designated B1, is shared among a subfamily of adhesion receptors that includes the receptors for laminin, collagens and tenascin. These receptors have been termed integrins. As each of the subunits has a cytoplasmic domain, both subunits possess the potential to interact separately or in combination with cytoplasmic proteins.
While not homologous with one another, the sequences of each of the subunit cytoplasmic domains have been shown to be highly conserved through evolution. For example, comparison of the sequence of the human fibronectin receptor ~ subunit with that of mouse shows that the cytoplasmic domains are identical. The cytoplasmic domains of the human, chicken and mouse B1 subunits are also ' ' '''' '.

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wosl/o275~ 2 0 6 4 8 1 8 PCT/US90/04662 identical. Furthermore, it has been shown that an antiserum against a synthetic peptide corresponding to the cytoplasmic domain of the chicken ~ subunit cross reacts with cell surface molecules from a number of evolutionarily lower species, including fungi. Such a high degree of conservation implies that these domains serve common roles from one species to another which are essential to receptor function.

lo It is believed that the fibronectin receptor and the other integrins function in integrating the extracellular matrix with the cytoskeletal framework. Indeed, the fibronectin receptor can be found in membrane-substratum adhesion sltes colocalizing with intracellular cytoskeletal proteins and extracellular fibronectin fibrils. However, the nature of the interaction between cytoplasmic domains of the integrins and other proteins has thus far remained elusive.

Because of the critical role which the integrins play in cell adhesion processes, there thus exists a need to identify the proteins that interact with these receptors and other components of the extracellular matrix so as to permit manipulation of these processes. The present invention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention is directed to a substantially purified protein, hereinafter termed "Fibulin," that interacts with the cytoplasmic domain of the B subunits shared by the fibronectin receptor and some other integrins. Fibulin has a molecular weight of about 100 kD under reducing conditions and binds to a peptide corresponding to the c~to~a~,mic domain of the fibronectin receptor in a divalent cation dependent and EDTA reversible manner.

.. . . ~ . .
- . - -, , ~ ' . ' 2 0~ ~ 8 ~/US90/04662 Human Fibulin has substantially the partial amino-terminal sequence D-V-L-L-E-A-C-C-A-D-G-H-R-M-A, and can have the amino acid sequences shown in Figures 3, 4 or 5.

This invention is further directed to isolates of nucleic aciq encoding Fibulin and to expression vectors harboring such nucleic acid in expressible form, to microorganism strains or cell cultures transformed with the vectors and to recombinantly produced Fibulin obtainable via expression of DNA encoding Fibulin in a transfected recombinant host system. Still further, the invention is directed towards antibodies reactive with Fibulin, be they monoclonal or polyclonal, and to nucleotide sequences capable of specifically hybridizing with nucleic acids encoding Fibulin.

BRIEF DESCRIPTION OF THE FIGURES

Figure l. Affinity chromatography of human placental extract on synthetic fibronectin receptor B subunit cytoplas~ic domain peptide-Sepharose. Lanes 1-8 represent SDS-PAGE analysis of fractions eluted from the affinity column using a cation free-buffer containing 20 mM EDTA.
Aliquots from each l/4 column volume fraction were -;25 electrophoresed on a 10% polyacrylamide gel in the presence ;of the reducing agent B-mercaptoethanol. Following electrophoresis the gel was stained with Coomassie Blue.
Molecular weight markers are indicated on the right in kilodaltons.
, . .
Figure 2. Binding of fibronectin receptor to Fibulin.
In A, varying concentrations of fibronectin receptor were incubated with microtiter wells coated with Fibulin (closed circles) bovine cardiac ~-actinin (open triangles) and bovine serum albumin (open circles). In B, the effect of EDTA on fibronectin receptor binding to Fibulin was examined. Bound receptor was detected by ELISA using a .

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WO9l/OZ7~ ~ 0 6 ~ 8 1 ~ 4 PCT/US90/04662 rabbit anti-fibronectin receptor serum. The data in each graph is representative of three experiments, each done in duplicate.

Figure 3 shows the complete nucleotide and predicted amino acid sequence for the A form of Fibulin. The putative signal peptide cleavage site is indicated by an arrow pointing upward. Protein sequences of the amino-terminus and of three tryptic peptides of Fibulin are indicated by solid lines. Amino acid residues beneath the predicted amino acid sequence indicate differences between the cDNA deduced sequence and those determined form protein sequencing. Potential N-linked carbohydrate attachment sites are indicated by solid squares. The site where the three types of Fibulin cDNAs diverge is indicated by an arrow pointing downward between nucleotides 1707 and 1708.

Figure 4 shows the sequence of the alternatively spliced segments from Fibulin B beginning at nucleotide 1708. Sequences of B that overlap with those of form A
(bases 1-1707) are not shown. Putative polyadenylation signal sequences are boxed.

Figure 5 shows the sequence of the alternatively spliced segments from Fibulin C beginning at nucleotide 1708. Sequences of C that overlap with those of form A
(bases 1-1707) are not shown. Putative polyadenylation signal sequences are boxed.

A novel protein that binds to an affinity matrix prepared from a synthetic peptide corresponding to the cytoplasmic domain of the fibronectin receptor B1 subunit has been isolated and characterized. This protein, which is termed "Fibulin," is a component of the extracellular . . . ~: :

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WO91/0275~ 2 ~ ~ ~ 8 ~ ~ PCT/US90/04662 matrix and participates in cell adhesion processes. Such adhesion processes include interactions with the fibronectin receptor and other adhesion receptors that share the B~ subunit.

Fibulin exhibits the following features: 1) it binds to an affinity matrix made from a integrin receptor B1 subunit cytoplasmic domain coupled to a solid support, such as Sepharose and is specifically eluted with the peptide or with EDTA; 2) it does not detectably bind to a column made with an ~ subunit cytoplasmic peptide, nor is it eluted from the B1 subunit column with the ~ subunit peptide; 3) native fibronectin receptor interacts with Fibulin in a concentration-dependent manner in an ln vitro binding assay; and 4) indirect immunofluorescent staining of cultured cells grown on fibronectin reveals that Fibulin colocalizPs with the fibronectin receptor ~ subunit in focal contact-like sites as well as areas of extended substrate contact.
.
As used herein, the term "Fibulin" refers to a protein having substantially the amino-terminal amino acid sequence in humans presented in Example V and the additional amino acid sequence encoded by the nucleic acid sequence of Figures 3, 4 or 5. Fibulin binds to synthetic peptides corresponding to the cytoplasmic domains of the Bl subunit of integrins and the cytoplasmic domains of the B2 and B3 subunits as well, and to the native fibronectin receptor, according to the binding described herein below. Fibulin refers both to protein native to mammalian tissue as well as to proteins produced by cell culture or recombinant methods. It will be understood that Fibulin includes the encoded polypeptide chain, post-translational modifications to the polypeptide and limited modifications to the protein including fragments which are antigenically or biologically active. For example, the protein may vary somewhat between species and natural allelic variations can occur from . .: - . . . . . . .

WO91/027S5 2 0 ~ PCT/US90/04662 individual to individual. These variations can include deletions, substitutions, insertions, inversions or additions of amino acids. These modifications may be deliberate, as through slte dlrected mutagenesis, or may be accidental, such as through mutation of the DNA of hosts which are Fibulin producers. The DNA encoding the protein can be alternatively spliced to yield different forms of the protein such as those differences shown in Figures 3, 4 and 5.

As is well-known in the art, proteins can exist in neutral form or in the form of basic or acid addition salts depending on their mode of preparation or environment, if ; in solution. Fibulin, in particular, may be found in the form of its acid addition salts, involving the free amino groups, or its basic salts, formed with free carboxyls.

Moreover, the protein may be modified by combination with other biological materials, such as lipids and saccharides, or by side chain modification, such as acetylation of amino groups, phosphorylation of hydroxyl side chains or oxidation of sulfhydryl groups. In addition, the location and degree of glycosylation will depend on the nature of the host cellular environment. All of these modifications are included in Fibulin.
' The term "nucleic acid which encodes for Fibulin" as used herein refers to the primary nucleotide sequence of a gene or cDNA encoding the amino acid sequence Fibulin, as defined above. An example is the sequence presented in Figures 3, 4 and 5. The gene may or may not be expressed in the native host. If it is not expressed in the native host, it may still be capable of being manipulated through recombinant techniques to effect expression in a foreign host. The term refers both to the precise nucleotide sequence of a gene found in a mammalian host as well as modified genes which still code for a Fibulin polypeptide ':

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W091tO275~ 2 0 ~ ~ 8 ~ 8 PCT/US9o,04662 having biological activity. The gene may exist as a single contiguous sequence or may because of intervening sequences and the like, exist as two or more discontinuous sequences, which are nonetheless transcribed ln vivo to ultimately effect the biosynthesis of a protein substantially equivalent to that defined as Fibulin, above.

The term "substantially pure," when used herein to describe the state of Fibulin, means substantially free of non-Fibulin proteins or other materials normally associated with Fibulin in its natural environment. More than one form or variant protein may be present, however, in the "substantially purified" form.

As used herein, "fibronectin receptor" refers to that receptor described in Pytela, et al., Cell 40:191-198 (1985) and Argraves, et al., J. Cell Biol. 105:1153 (1987), both of which are incorporated herein by reference.

Amino acids are identified herein by their standard abbreviations, as follows:

WO91/02755 PCT/US90/04662 _~
'~64~18 8 Amino Acid Sym~ol Alanine A
Arginine R
Aspartic acid D
Asparagine N
Cysteine C
lo Glutamine Q
Glutamic acid E
Glycine G
Histidine H
Isoleucine Leucine L
Lysine K
Methionine M
Phenylalanine F
Proline p Serine S
Threonine T
Tryptophan W
Tyrosine y Valine V
The invention provides substantially purified Fibulin and substantially purified Fibulin which has an apparent molecular weight under reducing conditions of 100 kD and which binds to the cytoplasmic domain of the B1 subunit of integrin adhesion receptors in a cation dependent, EDTA
reversible manner.

The invention provides a substantially purified polypeptide, wherein said polypeptide has substantially the partial amino terminal sequence:
D-V-L-L-E-A-C-C-A-D-G-H-R-M-A. The invention provides a protein having an amino acid sequence substantially the same as that encoded by the nucleic acid sequence of Figures 3, 4 or 5.
;

The invention provides antibodies reactive with Fibulin or antigenic determinants thereof. The invention provides antibodies reactive with Fibulin or antigenic determinants thereof, wherein said antibodies are ' ' ~ ' W091/02755 2 a~ 8l 8 pcT~usgo~o4662 monoclonal. The invention provides antibodies reactive with Fibulin or antigenic determin~nts thereof, wherein said antibodies are polyclonal.

The invention provides an isolated nucleic acid which encodes Fibulin and nucleic acid complementary to nucleic acid encoding Fibulin. The invention provides the isolated nucleic acid which encodes Fibulin, wherein the nucleic acid has substantially the sequence as that shown for base 10 pairs 1 through 1707 in Figures 3, 4 or 5.

The invention provides the nucleic acid which encodes Fibulin, wherein the sequence further comprises substantially the sequence as that shown for the base pairs 15 beginning at 1708 and extending to the 3' terminus in -Figures 3, 4 or 5. The invention provides the nucleic acid of Fibulin wherein said nucleic acid is cDNA. ~-The invention provides a recombinant DNA cloning vector operatively harboring a DNA sequence coding for Fibulin. The invention provides a host'transformed by the cloning vector harboring Fibulin. The invention provides a recombinant DNA sequence effective, in compatible host cells, of effecting the expression of Fibulin DNA. The invention provides a process comprising expressing DNA
encoding Fibulin in a host cell.

The invention provides a method for purifying Fibulin from a Fibulin-containing material comprising the steps of:
immobilizing a peptide substantially comprising the cytoplasmic domain of B1 integrin subunit on a solid support; contacting said Fibulin-containing material with said immobilized cytoplasmic domain of B1 integrin; removing material not bound to said immobilized cytoplasmic domain of the B1 integrin subunit; and recovering material bound to said immobilized cytoplasmic domain, wherein said recovered material is substantially purified Fibulin.

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The invention provides a method of characterizing the extracellular matrix as to the presence of Fibulin, comprising the steps of: contacting said extracellular matrix with antibodies reactive with Fibulin or antigenic determinants thereofi and determining whether said antibodies bound to said extracellular matrix.

The invention provides substantially purified Fibulin having biotin attached thereto.

The invention provides a method of targeting a moiety to the extracellular matrix of an organism comprising:
attaching said moiety to Fibulin, to form a Fibulin complex; and administering said Fibulin complex to said organism. The invention provides the method of targeting a moiety to the extracellular matrix wherein said moiety is biotin.

The invention provides a method of purifying Fibulin from a Fibulin-containing material comprising the steps of:
immobilizing a lectin on a solid support; contacting said Fibulin-containing material with said immobilized iectin;
removing material not bound to said immobilized lectin; and recovering material bound to said immobilized lectin, wherein said recovered material is substantially purified Fibulin. The invention provides the method of purifying Fibulin on said immobilized lectin wherein said lectin is wheat germ agglutinin.

Fibulin can be purified from Fibulin-containing material, such as a tissue extract, by contacting the Fibulin-containing material with a solid support, such as beads, to which is attached the cytoplasmic domain of integrin, or a lectin such as wheat germ agglutinin.
Fibulin selectively binds to the cytoplasmic domain or lectin.

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Fibulin was isolated from an extract of human placental tissue on an affinity column. A synthetic peptide corresponding to the cytoplasmic domain of the fibronectin receptor B subunit (residues 762-798, Argraves et al., J. Cell Biol. 105:1153 (1987), which is incorporated herein by reference), was coupled to Sepharose ; and used as an affinity matrix in the presence of buffers containing divalent cations and octylglucoside to select putative binding proteins from an extract of human lo placenta.

Elution with an EDTA-containing buffer released the Fibulin protein which, when reduced and electrophoresed on SDS-PAGE, had an apparent molecular weight of lOO,OoO
daltons (100 kd) (Fig. 1). Following the EDTA elution, no additional Fibulin was released from the column by 8 M urea suggesting that quantitative removal was achieved by the EDTA. Peptides corresponding to the cytoplasmic domain of the B1 subunit also are capable of partially eluting Fibulin from the column. However, elution using a synthetic peptide corresponding to the ~ subunit cytoplasmic domain (residues 1028-1049, Argraves et al., supra) did not release Fibulin from the B peptide affinity matrix.
Additionally, Fibulin did not bind to a column prepared with the synthetic ~ subunit cytoplasmic peptide.
.
When the affinity-selected protein was electrophoresed on SDS-PAGE under non-reducing conditions, two polypeptide bands with mobilities corresponding to molecular weights of approximately 80 kd and 200 kd were seen. These polypeptide bands were excised from gels and the protein isolated by electroelution. When the eluted proteins were re-electrophoresed on SDS-PAGE under reducing conditions, both migrated with an apparent molecular weight of 100 kd.
The difference in the reduced and non-reduced monomer mobility on SDS-PAGE is likely due to intramolecular disulfide bonding making the non-reduced form more compact . . . .

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WO91/0275; 2 0 ~ CT/US90~04662 --and thus having higher electrophoretic mobility. The~200 kd form may be a disulfide linked dimer or trimer.

Amino acid sequencing of Fibulin indicated the amino-5 terminal sequence to be: D-V-L-L-E-A-S-X-A-D-G-S-H-M-A. X -~
indicates a position where no amino acid determination could be made. Subsequent cloning and nucleotide sequencing confirmed most of the residues and indicated a cysteine residue at the X position and moreover, indicated that the seventh position serine is also a cysteine, that the eleventh position is histidine and that the twelfth position is arginine. It will be appreciated by those skilled in the art that amino acid sequencing can, for various reasons, yield equivocal results, particularly where the amount of protein available is small. It is believed that the amino acid sequence deduced from the nucleotide sequence is substantially correct.
~ ' The amino acid sequence was used to search the Protein Identification Resource database. No protein present in the database was found to share this sequence or to be closely similar, indicating that Fibulin does not correspond to any previously sequenced protein.

The cDNAs obtained showed that there exists at least three forms of Fibulin (designated A, B, and C) encoded by three transcripts likely derived from a common pre-mRNA.
The nucleotide and predicted amino acid sequence of the Fibulin forms A, B and C are shown in figures 3, 4 and 5, respectively. The fact that the Fibulin preparations seem only to have a single polypeptide indicates that predominantly one form is being isolated or expressed at high levels. Another puzzling issue has to do with the disparity between the molecular weight of Fibulin estimated from SDS-PAGE and that determined from cDNA. The polypeptides (minus signal peptides) predicted from the nucleotide sequences of the three cDNAs have molecular ,, : - , , .. ... .

2~6~8~ 8 weights of 58,670, 62,561, and 71,551 daltons. These values are not in agreement with Fibulin's apparent molecular weight of lOO kd obtained from SDS-PAGE.
Carbohydrate analysis indicated that N-linked glycosylation -5 only accounts for approximately 4-5 kd of the molecular mass of the lOO kd polypeptide. Other types of substitution, such as O-glycosylation, may account for the remaining difference. Overestimation of molecular weight by SDS-PAGE has been reported for a number of proteins rich 10 in negatively charged amino acids and having low isoelectric point (pI) values. Fibulins A, B and C have an average content of aspartic and glutamic acid residues of 13.5% and average calculated pI of the polypeptide chain of 4.7. It is therefore possible that anomalous 15 electrophoretic behavior of Fibulin on SDS-PAGE results in an overestimation of its size.

Once the amino terminal sequence of a protein has been determined and antibodies to the protein obtained, the gene 20 of interest can be identified and isolated from a cDNA or genomic library. Once isolated, the gene or cDNA can be subcloned into an expression vector and introduced into host cells which allow the transcription of the gene and translation into the final protein product. The 25 recombinant protein can be purified from the cells by methods known to those skilled in the art, as, for example, by affinity purification using an antibody specific to the Oprotein of interest. See generally, DNA CLONING: VOLUME I
& II (D.M. Glover ed. 1985); and ~aniatis et al, MOLECULAR
30 CLONING: A LABORATORY MANUAL, (1982) which are incorporated herein by reference.
. .:
While the affinity chromatography results indicated that Fibulin was able to interact with the synthetic B1 35 subunit cytoplasmic domain peptide, it was necessary to determine whether it was able to interact with native fibronectin receptor. A microtiter plate binding assay was ,, . . .,,, . . . .-...... .. . ~ , WO91/027~5 PCT/US90/04662 -~
~ 14 developed which examined the capacity of purified fibronectin receptor to bind to polystyrene microtiter plate wells coated with Fibulin. As documented in Figure 2, fibronectin receptor bound to Fibulin coated wells in a concentration-dependent manner. Control experiments showed that under similar conditions the receptor did not bind to either bovine cardiac ~-actinin or bovine serum albumin coated wells.

To determine if the ln vitro interaction between the receptor and Fibulin was divalent cation-dependent, as was the interaction between Fibulin and the synthetic cytoplasmic domain affinity matrix, divalent cation levels were modulated with EDTA. As shown in Figure 2, EDTA
indeed inhibited the fibronectin receptor binding to Fibulin coated wells. To ensure that the EDTA was not -causing removal of Fibulin from the microtiter plate during the assay an ELISA was performed on the protein coating following treatment with EDTA. This assay showed that the amount of Fibulin coating the wells remained unchanged by EDTA at the concentrations used in the binding assay.
These results indicate that native fibronectin receptor is capable of directly interacting with Fibulin. Furthermore, the interaction is dependent on the presence of divalent cations.
:; .
The fact that EDTA could cause the disassociation between Fibulin and both the synthetic receptor cytoplasmic domain and the native fibronectin receptor prompted determination of whether Fibulin was capable of binding divalent cations. Nitrocellulose filters containing Fibulin electrophoretically transferred from SDS-PAGE were incubated with radioactive 45Ca2. The radioactive calcium bound specifically to Fibulin. This result indicates that Fibulin is indeed a calcium binding protein.

Analysis of the predicted amino acid sequences ,: ' : -' ~' ' ' ' ':
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2 0 ~ p8cr WO91/02755 ~ /US90/04662 (Figures 3 through 5) indicated no sequence homologous to the consensus divalent cation binding sequences of proteins such as calmodulin, troponin C and parvalbumin. The analysis did reveal, however, the presence of four potential asparagine hydroxylation sites, CX(D/N)(~)4(F/Y~XCXC, (Stenflo et al., J. Biol. Chem.
263:21-24, 1988, which is incorporated herein by reference) embodied within EGF-like repeats 5-8. A total of nine EGF-like repeats exist in Fibulins A, B and C. EGF domains containing B-hydroxylated residu~s have been implicated in calcium-binding and are found in numerous proteins including: the vitamin K-dependent blood coagulation proteins, complement protein Clr, low density lipoprotein receptor and thrombomodulin.

In order to determine the cellular localization of Fibulin, human gingival fibroblasts were grown on fibronectin coated surfaces and examined by indirect immunofluorescent microscopy using rabbit anti-lO0 kd protein serum and fluorochrome conjugated anti-rabbit IgG
serum. Fibulin antiserum specificity was confirmed by immunoprecipitation analysis. The antiserum precipitated a single protein with a molecular weight corresponding to Fibulin from a Triton X-100 extract of 35S-cysteine labeled gingival fibroblasts. Fluorescent staining of Fibulin was very prominent when the cells were permeabilized with detergent. Most non-permeabilized cells showed no staining for Fibulin, however, sparse staining was noted on the margins of some cells. Similar levels of peripheral staining of non-permeabilized cells was also seen using 3Q antibodies to ~-actinin, a known intracellular protein (Lazarides and Burridge, Cell 6:289 (1975), which is incorporated herein by reference). It was therefore concluded that Fibulin is an intracellular protein and that the observed non-permeabilized staining may be artifactually related to fixation. Some Fibulin does, however, exist extracellularly.

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The immunostaining of permeabilized cells revealed Fibulin to be distributed in striated or streak-like accumulations throughout the cell. These streaks were most prominent in the periphery of the cell. No staining was apparent when pre-immune serum was used.

The distribution of Fibulin relative to the fibronectin receptor B~ subunit was examined by double-label immunofluorescent microscopy. Cells were stained with both a mouse monoclonal anti-fibronectin receptor B subunit antibody and the rabbit anti-100 kd protein serum. The receptor B subunit staining that was observed was similar to what others have reported using B subunit specific antibodies (Marcantonio and Hynes, J. Cell Biol. 106:1765 (1988), which is incorporated herein by reference). The fibronectin receptor B subunit accumulated in focal contact-like sites in peripheral regions of the cell. The staining took the form of streaks of fluorescence typical of extended substrate contacts. Overlapping Fibulin staining was seen in the focal contact-like sites also taking the form of streaks of fluorescence, however, the streaks were finer and more elongated. The codistribution suggests that there is an ln vivo association between Fibulin and the receptor B subunit.

The above observations were based on immunofluorescent staining studies done on fibroblasts grown for 4-6 hours on fibronectin coated surfaces. However, when cells were stained with a monoclonal antibody to fibronectin or one recognizing a presumed extracellular determinant of integrin ~1 subunit, no specific staining was found unless the cells were permeablized. Evidently, in the absence of permeablizing agents, the close association between the cell and the substratum prevents access of antibodies to sites where fibronectin and integrin are accumulating.
Therefore, the ability to see staining only after permeablization may be a misleading indicator that the ~. .
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W O 91/02755 2 ~ 6 4 8 1 ~ PC~r/USgO/04662 17 :
target antigen is an intracellular protein.

When immunofluorescent staining studies were extended to periods beyond 4-6 hours, staining of Fibulin in the absence of permeablization was seen. With progressive culture time, Fibulin was found to accumulate extracellularly into extensive fibrillar patterns resembling the pattern of accumulation of fibronectin.
Using pulse-chase labeling and immunoprecipitation lo analyses, it was established that Fibulin was indeed a secreted protein. Furthermore, lectin affinity chromatography and N-glycosidase digestion showed that Fibulin was a glycoprotein containing N-linked carbohydrate. These findings were supported by the results of cDNA cloning which showed the predicted amino acid sequence of Fibulin to have a signal sequence and 3 potential N-glycosylation sites. The presence of the repeated EGF-like motif is yet another feature not found in cytoplasmic proteins but common to a number of extracellular matrix, plasma, and membrane proteins (Engel, FEBS Letters ~51:1-7, 1989, which is incorporated herein by reference). Taken together the findings are consistent with Fibulin being an extracellular matrix protein rather than a cytoplasmic protein. The significance of the fact that Fibulin can be purified by affinity chromatography on the putative cytoplasmic domain of the B1 subunit remains to be explained. However, Fibulin being an extracellular matrix component is beneficial for targeting of moieties such as biotin or other macromolecules to the extracellular matrix. Such targeting can be accomplished by attaching the moiety to Fibulin to form a complex and adding the complex to an extracellular matrix. The complex can be administred in vitro or in vivo.

35The dissociation by EDTA of the interaction between Fibulin and the native fibronectin receptor as well as Fibulin and the synthetic receptor cytoplasmic domain - - - . . ~ -. . . : . : -.. . .. ..
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WO91/027~ PCT/US90/04662 affinity matrix by EDTA indicates a divalent cation requirement for the interaction. Calcium is implicated as a required divalent cation since Fibulin was found to bind radioactive calcium. Calcium regulation has been postulated in the interaction of actin with itself as well as with actin binding proteins. It is then possible that the calcium regulation not only affects the dynamics of microfilament assembly but also the interaction of such filaments with adhesion receptors in the plasma membrane.
Fibulin can be used to manipulate such interactions since it binds to the cytoplasmic domain of the B1 subunit of adhesion receptors.

The physical properties of Fibulin clearly distinguish it from talin, a 225 kd protein (Burridge and Connell, J.
Cell Biol. 97:369 (1983~; Molony et al., J. siol. Chem.
262:7790 (1987), both of which are incorporated herein by reference) that has been shown to bind to chicken integrins in vitro (Horwitz et al., Nature, 320:931 (1986), which is incorporated herein by reference) and to colocalize with integrins (Burns et al, Proc. Natl. Acad. Sci. U.S.A., 85:497 (1988), which is incorporated herein by reference), ln vivo. No protein bands corresponding to intact talin or its proteolytic breakdown products (O'Halloran et al., Nature 317:449-451 (1985), which is incorporated herein by reference) were apparent in the SDS-PAGE profile of fractions eluted from the affinity matrix. If talin binds to the B subunit the binding is too weak to be apparent under the conditions we have used. Alternatively, the talin binding may require the ~ subunit.

The physical properties of Fibulin also differ from those of other known focal-contact associated and microfilament associated proteins (Geiger, B., Biochem.
Biophys. Acta, 739:305 (1983); Weeds, Nature 296:811 (1982), both of which are incorporated herein by reference). Two such proteins, gelsolin and ~-actinin, . . .:

' , .. :, ::, . i WO91/02755 2 ~ ~ ~ 8 1~ PCT/US90/04662 posses some characteristics common to Fibulin. These characteristics include similar molecular weight, ability to bind calcium, and cellular localization. No similarity was found between the amino-terminal sequence of human gelsolin (Kwiatkowski et al., Nature 323:455 (1986), which is incorporated herein by reference) and that determined for Fibulin. Likewise, a comparison of the amino-terminal sequence of Fibulin with that for chicken skeletal muscle ~-actin (Arimura et al., Eur. J. Biochem. 177:649 (1988), which is incorporated herein by reference) showed no similarity. Immunologically, no relationship between ~-actinin and Fibulin was found. Anti-~-actinin serum reactive with human muscle and non-muscle isoforms showed no reactivity with either reduced or non-reduced Fibulin transferred to nitrocellulose from SDS-PAGE. Furthermore, anti-Fibulin serum showed no reactivity with bovine cardiac ~-actinin in immunoblotting.

When the amino terminal sequence of Fibulin was used to search the NBRF protein database, no protein present in the database was found to share the sequence or to be closely similar. In addition, sequences have been determined from three tryptic fragments of Fibulin. When these sequences were used to search the protein database no matches were found. The evidence thus far obtained indicates that Fibulin is a new molecule that may serve as a link between the fibronectin receptor B subunit and cytoplasmic components, perhaps the cytoskeleton.

The B subunit of the fibronectin receptor (B1 subunit) has been shown to be sh~red with at least six other integrin receptors (Hemler et al., J. Biol. Chem. 263:17660 (1987), J. Biol Chem. 262:3300 (1988), both of which are incorporated herein by reference). Several other members of this group are known to be receptors for the extracellular matrix molecules laminin and collagen. By interacting with all of the receptors possessing the B1 .. -: - :. . .
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WO91/~275~ 2 0 ~ ~ 818 PCT/US90/04662 subunit, Fibulin can be used to manipulate adhesion of cells at least to fibronectin, collagen, laminin, and possibly to other proteins as well.

Either native or synthetic Fibulin, or peptides corresponding to antigenic determinants thereof, can be used to produce antibodies, either polyclonal or monoclonal. If polyclonal antibodies are desired, antigen is used to immunize a selected mammal (for example, mouse, rabbit, goat, horse, etc.) and serum from the immunized animal is later collected and treated according to known procedures. Antisera containing polyclonal antibodies to a variety of antigens in addition to those to the antigen of interest can be made substantially free of antibodies which are not antigen specific by passing the composition through a column to which non-antigen protein has been bound. After washing, antibodies to the non-specific proteins will bind to the column, whereas antibodies to antigen of interest elute in the flow through. Monoclonal antibodies can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by fusing myelomas and lymphocytes to form hybridomas is well known. Such cells are screened to determine whether they secrete the desired antibodies, and can then be grown either in culture or in peritoneal cavity of a mammal. Antibodies can be recovered from the supernatant or ascites fluid. Immortal, antibody producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.
See, for example, M. Schrier, et al., HYBRIDOMA TECHNIQUES
(1980); Hammerling, et al., MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS (1981); Kennet, et al., MONOCLONAL
ANTIBODIES (1980); Harlow and Lane, ANTIBODIES: A
LABORATORY MANUAL, Cold Spring Harbor (1988), all of which are incorporated herein by reference. Such antibodies can be used to characterize an extracellular matrix as to the . ~ . , , ,, . :, - :

~ .
.' '~
, ' , ~ , . , ,' ~ ., ,~ ' ' ` ,,' , WO9l/OZ755 ~ 6 4 ~ 1~ PCT/US90/04662 presence of Fibulin.

Because of the integral role of the cell adhesion system in mediating attachment, migration, growth, metastasis and differentiation of cells and tissues, Fibulin and antibodies reactive with Fibulin or antigenic determinants thereof, has important diagnostic and therapeutic utilities for both normal and abnormal conditions. In addition, Fibulin can be used to target lo moieties to the extracellular matrix, by attaching such moieties to Fibulin.

The following examples are intended to illustrate but not limit the invention.

EXAMPLE I
AFFINITY CHROMATOGRAPHY

The following peptide, corresponding to residues 762-798 of the fibronectin receptor B subunit (Argraves et al., 20 J. Cell Biol., 105:1183, (1987a)), was synthesized using an Applied Biosystems model 430A, according to the manufacturer's instructions:
.
E-F-A-X-F-E-K-E-K-M-N-A-K-W-D-T-G-E-N-P-I-Y-K-S-A-V-T-T-V-V-N-P-K-Y-E-G-K

An affinity matrix was prepared by coupling the peptide to cyanogen-bromide-activated Sepharose (Pharmacia, Piscataway, NJ), according to the manufacturer's instructions. The resulting matrix contained approximately 10 mg peptide per ml of Sepharose.
:
Human placental tissue (100 g, ground) was washed with two volumes of 0.005% digitonin, Sigma Chemical Co., St.
Louis, MO, 1 mM CaCl2, 1 mM MgCl2, TBS (150 mM NaCl, 50 mM
Tris-HCl pH 7.4), 1 mM phenylmethylsulfonylfluoride (PMSF).

: . . - . . , -~ .... ;,.. -. ~ : - ~ . , .. . ~ . .... .
. : - : , ' ' ~ ''' ' . ' ': ' ''~
~, :

, . , ... ~ .
.. . . .. ~ ,. . . .

:

WO91/02755 2~ 1 8 PCT/US90/04662 After centrifugation at 2600 X g for 15 minutes, the pelleted tissue was extracted with 100 ml of 50 mM octyl-B-D-glucoside (Calbiochem, La Jolla, CA), 1 mM CaCl2, TBS, 1 mM PMSF according to the procedure of Pytela, et al.
Methods Enzymol. 144:475 (1987). Extracts clarified by centrifugation at 2600 X g were first applied to a column of plain Sepharose CL-4B (15 ml) and then to the synthetic peptide affinity matrix (7 ml). The column was washed with 8 column volumes of 25 mM octyl-~-D-glucoside, 1 mM CaC12, 1 mM MgCl2, TBS, 1 mM PMSF (wash buffer). Elution was achieved using 2 column volumes of cation free wash buffer containing 20 mM EDTA. Samples from each 1/4 column volume fraction were analyzed by SDS-PAGE (Laemmli, Nature, 227:680 (1970), which is incorporated herein by reference) and protein bands stained using Coomassie Brilliant Blue R-250. Typically the yield was 200-300 ~g of Fibulin from 100 g of placental tissue. These results (Figure 1) indicate that Fibulin interacts with the synthetic cytoplasmic domain affinity matrix in a divalent cation dependent manner. Other experiments showed that elution with buffer containing the synthetic peptide residues 762-798 of the ~1 subunit, or 8 M urea also released Fibulin from the affinity matrix.

25Elution using a synthetic peptide corresponding to the subunit cytoplasmic domain (residues 1028-1049, Argraves et al., su~ra) did not release Fibulin from the ~ peptide affinity matrix. Additionally, Fibulin did not bind to a column prepared with the synthetic ~ subunit cytoplasmic peptide.

EXAMPLE II
POLYCLONAL ANTIBODY PRODUCTION

35Affinity chromatography purified Fibulin (100 ~g in 0.5 ml TBS) was mixed with an equal volume of Freund's complete adjuvant, emulsified, and used to immunize a New , ,, . ~, - , , . ;: ;. -~ : .

~ " : ' '' ' ~:
', . ' 20~8~8 Zealand White femal~ rabbit. Booster injections of the same dose of protein emulsified in Freund's incomplete adjuvant were made. The rabbit was boosted 3 weeks following the immunization, then bled 8 days later.
Subsequent booster injections were administered after 1 month and bleeding done as before (this cycle has been repeated several times). The titer of the serum was monitored by enzyme-linked immunosorbent assays (ELISA, Engvall and Pearlman, J. Immunol. 109:129 (1972), which is lo incorporated herein by reference) and immunoblot analysis.
This antiserum is designated 1323. As a precaution, the serum used in the immunofluorescent experiments of Example X was absorbed on columns of human fibronectin and fibronectin receptor coupled to Sepharose.

For immunoadsorbtion and ELISA of Fibulin, the mouse monoclonal antibody 5D12/H7 was used. This hybridoma cell line was produced by fusion of immune mouse spleen cells with myeloma X63Ag8.653 cells according to published methods (Ruoslahti et al., Meth. Enzymol. 84:3-19, 1982, which is incorporated herein by reference). 5D12/H7 reacts specifically with Fibulin in ELISA, immunoprecipitation and in immunoblotting under both reducing and non-reducing conditions.

EXAMPLE III
MONOCLONAL ANTIBODY PRODUCTION

Balb/c mice were injected intraperitoneally with approximately 50 ~g of Fibulin emulsified in Freund's complete adjuvant (1:1 vol/vol). Boosts were done in the same fashion 4 weeks later with Fibulin emulsified in Freund's incomplete adjuvant. Five days later splenectomy was performed. Fusions were performed essentially according to protocols described in (Hessle and Engvall J.
Cell Biol. 259:3955-3961 (1984) and Ruoslahti et al., Meths. Enzymol. 84:3-19 (1982), both of which are . . . - - , ... ~ .

. ~ ~, ,- '.
. ' ' . .
.' . '' ~ . . -: . . .. . :. . ...

WO91/027~5 PCT/US90/04662 ~
;~6~818 24 incorporated herein by reference) using murine myeloma line X63-Ag8.563. Mouse monoclonal anti-human fibronectin which does not cross-react with bovine fibronectin was purchased from Telios Pharmaceuticals, San Diego, CA.

AMINO-TERMINAL SEQUENCE ANALYSIS

Affinity chromatography-isolated Fibulin was subjected to SDS PAGE in a 15% acrylamide gel under reducing conditions. Following electrophoresis the gel was stained with coomassie Blue for 5 minutes and destained just until the lOO kd band was apparent. The band was excised from the gel and the protein electroeluted (Hunkapiller et al., Meth. Enzymol. 91:227 (1983), which is incorporated herein by reference). SDS was removed from the electroeluted protein according to the procedure of (Konigsberg and Henderson, Meth. Enzymol. 91:754 (1983), which is incorporated herein by reference) and approximately lOO
pmol was subjected to Edman degradation using an Applied Biosystems (Foster City, CA) model 477A protein sequencer with an on-line Applied Biosystems model 130 phenylthiohydantoin analyzer.

The following sequence was determined:

X could not be conclusively identified.

The amino-terminal sequence that was obtained was used to search the Protein Identification Resource (1~88) Protein Sequence Database (National Biomedical Research Foundation (NBRF), Washington, DC). No homologous proteins were identified.

. .. . ~ ~

206~8~8 . ...

EXAMPLE V
ISOLATION AND CHARACTERIZATION OF cDNA CLONES

A polyclonal antiserum prepared against Fibulin was used to immunologically screen a lambda gtll human placental cDNA library essentially as described previously (Argraves et al., J. Cell Biol., 105:1183 (1987), which is incorporated herein by reference). Selected clones were shown to express insert encoded protein reactive with antibodies affinity selected on Fibulin-Sepharose. cDNA
inserts from 3 of these clones were isolated and subcloned into the sequencing ~ector ml3mpl9. The cDNA inserts were sequenced by the dideoxy chain termination method (Sanger et al., Proc. Natl. Acad. Sci. 74:5463-5467 (1977), which is incorporated herein by reference). The identity of the cDNAs was confirmed by the fact that they could be shown to encode Fibulin amino acid sequences determined by protein sequence analysis.

The partial Fibulin cDNA nucleotide sequence and deduced amino acid sequence are as follows:

D V L L E A C C A D G H R M A T H
S X S H
:
Differences between the sequence obtained from amino acid sequence analysis and deduced sequence are indicated below the deduced amino acid sequence.

Further, immunological screening of a placental cDNA
library resulted in the isolation of 7 related clones. As individual cDNAs were sequenced it was found that they - could be categorized into three types (A, ~, and C) as shown in Figures 3, 4 and 5, respectively. The nucleotide sequence of all three types of cDNAs were identical from their 5' ends to a divergence point at position 1707, after ' -W09]/Ot755 2 ~ ~ 4~ 1~ 26 PCT/US90/04662 which they were distinct through to the poly(A) tail. The categorization was therefore based on the sequence following the divergence point.

Polypeptides of 566, 601 and 683 amino acid residues are encoded by the type A (Figure 3), B (Figure 4) and C
(Figure 5) cDNAs, respectively. These polypeptides have in common the first 566 amino acid residues. The alternative B and C cDNA segments encode differing polypeptide elements that add 35 and 117 residues to the 566 residue protein.
The amino acid sequence deduced from the cDNAs was found to contain the sequences determined from the protein sequencing of Fibulin including the amino-terminal sequence and three sequences derived from tryptic fragments of Fibulin (Figure 3). These findings additionally confirmed that the immunologically identified cDNAs indeed corresponded to Fibulin. Preceding the amino-terminal sequence in the deduced type A, B and C sequences is a 29 residue hydrophobic leader sequence that has features consistent with it being a signal peptide. Three potential N-linked glycosylation sites (N-X-S/T) occur in each of the deduced sequences.

The three forms of Fibulin are rich in cysteine (approximately 11 mol ~), containing 69, 70 and 72 residues for the A, B and C forms, respectively. Analysis of the sequence with respect to the number and spacing of cysteine residues revealed the presence of two types of repeat motifs (designated type I and II) that each share homology with elements from specific proteins found in the database.

The type I motif has a consensus sequence CC(X)12C(X)9 C(X)6CC, and is repeated twice. Separating the two is an imperfect form of this motif that lacks two cysteines. A
computer aided search of the protein database for sequences containing the type I motif or slight variations thereof - revealed that CC(X)12C(X)1112C(X)6CC is found in complement ~. :

2~81 8 W091/0275~ PCT/US90/04662 - component anaphylatoxins C3a, C~a and C5a. The inverse pattern, CC(X)6C~X)1~2C(X)~2CC, is found in the three members of the albumin gene family which include albumin, vitamin D-binding protein and ~-fetoprotein. The homology findings suggest that the overall disulfide stabilized loop structure may be conserved between Fibulin and these other proteins even though similarity between residues other than cysteine in the pattern is unremarkable.

The type II motif of Fi~ulin is related to the repeats found in epidermal growth factor precursor as well as a number of extracellular matrix proteins. This 6 cysteine motif is repeated consecutively nine times in the sequence of Fibulin A, B and C. Four of the nine type II repeats (2-4, and 9) differ from the typical EGF-like motif in that they have a 4-6 residue insertion between cysteines 4 and 5, instead of the usual single residue separating the two.
The ninth type II repeat of Fibulin A is imperfect in that it lacks a cysteine in the sixth position of the motif while Fibulins B and C both have cysteine residues in the vicinity, but the spacing of these is not conserved relative to the other repeats. Embodied within the four of ; the nine type II repeats (5-8) is consensus sequence for aspartic acid and asparagine hydorxylation. The seventh type II repeat contains a consensus 0-glycosylation sequence, CXCXPC, that is found in the EGF-like domains of coagulation factors, VII, IX, protein Z and thrombospondin.
Immediately following each type II repeat is a pentapeptide with a consensus sequence XD(I/V~(D/N)E. Separating the third type I repeat and the first type II repeat is a 33 residues segment with 36% (12) of the amino acids either aspartic or glutamic acid.

,,. - . - . - .. . .- ., - .: : , ., ~ ' : ' - . . :, ' ~ :
.: , , :
- . , ~ - . : , . - . - . . .
. . , . , : " . : . , , .: , . . :
.,, ~ : , . .
. '' ' : : .
., : , .: .
.

, ~:

WO91/02755 ~ 0 6 ~ PCT/US90/04662 EXAMPLE VI
FIsRoNECTIN RECEPTOR-FIBULIN IN VITRO BINDING ASSAYS

Microtiter plate wells (Becton Dickinson, Lincoln S Park, NJ) were coated with Fibulin at 1.0 ~g/ml in coating buffer (0.1 M sodium carbonate pH 8.5). Control wells were coated with bovine serum albumin (BSA) at 3 ~g/ml in coating buffer. Fibronectin receptor was added to wells at concentrations ranging from 20 ~g/ml to 0.01 ~g/ml in TBS, 0.5~ Tween-20, 1 mM CaCl2, 1 mM MgCl2 (TBS-Tween-cations).
The fibronectin receptor used in these assays was isolated by affinity chromatography on a column of the 120 kd cell binding fragment of fibronectin coupled to Sepharose (Pytela et al., Meth. Enzymol. 144:475-489 (1987), which is incorporated herein by reference) and further purified on wheat germ agglutinin-agarose (Vector Laboratories, Burlingame, CA). Following an 18 hour incubation at 4C
the fibronectin receptor containing solution was removed and the wells washed 3 times with TBS-Tween-cations.
Rabbit anti-fibronectin receptor serum (Arqraves et al., J.
Cell Biol. 105:1183-1190 (1987); Argraves, et al., J. Biol.
Chem. 261:12922-12924 (1987), both of which are incorporated herein by reference) absorbed previously on columns of Fibulin coupled to Sepharose and human serum proteins coupled to Sepharose was diluted 1:1500 and incubated with each well for 2 hours at 37C. The wells were then washed 3 times with TBS-Tween buffer containing cations. Goat anti-rabbit IgG alkaline phosphatase conjugate (BioRad, Richmond, CA) was added and incubated for 1 hour at 37C. The substrate paranitrophenyl phosphate (Sigma, St. Louis, M0), in 1 M diethanolamine, pH
9.8, 1 mM MqCl2, was added and at various time intervals the absorbance at 410 nm measured using a Dynatech (Model MR-600;) reader.

For assays that evaluated the effect of EDTA on the interaction between the fibronectin receptor and Fibulin, .. -. . - . .~. . ~ ~ :,: :. . ... . .

.. . - . ............ . . .
: '.

206~81 ~
Wo9l/o27ss PCT/US90/04662 wells coated with Fibulin (0.6 ~g/ml) were incubated with fixed concentrations of fibronectln receptor (20 ~g/ml in TBS-Tween-cations) in the presence of varying concentrations of EDTA (10 mM - 0.0046 mM). The receptor containing solutions were incubated for 18 hours at 4C
with Fibulin coated wells. Subsequent washing and immunological detection of bound receptor were performed as described above.

EXAMPLE VII
BINDING OF FIBULIN TO CYTOPLASMIC

In order to evaluate the potential of Fibulin to bind integrins other than the fibronectin receptor peptides corresponding to the cytoplasmic domains of the beta 2 and 3 subunits were synthesized on a Milligen peptide synthesizer (model 9050). These synthetic cytoplasmic domain peptides were coupled to Sepharose as described above and used as affinity matrices to select binding proteins from octylglucoside extracts of placenta. The columns were washed and eluted with an EDTA solution as described previously. Individual fractions were sepa~~ted by electrophoresis on SDS-polyacrylamide gels, staine~ ~ith ; 25 Coomassie and transferred electrophoretically to nitrocellulose. Protein transferred to nitrocellulose was immunologically stained with anti-Fibulin antibodies.

The results are presented in Table I. A yes indicates that the peptide affinity matrix bound Fibulin as indicated by elution of a Coomassie stainable band of similar molecular size to Fibulin as well as, cross reaction of the eluted protein with antibodies specific for Fibulin (1323 and 5D12/H7). A no indicates that the affinity matrix bound no detectable amounts of Fibulin as determined by Coomassie staining and immunoblotting. As can be seen from Table I, Fibulin was bound to peptides corresponding to the - : . : : ........ .
:'. , , : . , : . ' ~ ,~, . '. : ' . , - . .

- ~ .

WO9l/02755 2 0 ~ PCT/US90/04662 _ cytoplasmic domain of the B1 B2 and B3 subunits.

Table I
Fibulin Cytoplasmic domain Origin of Binding coupled to Sepharose Sequence (ref) Capacity (762) EFAKFEXEKMNAKWDTGEN BETA 1 (1) yes PIYKSAVTTVVNPKYEGK (798) (785) SAVTTW NPKYEGK (798) BETA 1 (1) no (762) EFAKFEKEKMNAKWDT (777) BETA 1 (1) yes 15 (734) EYRRFEKEKLKSQWNNDNP BETA 2 (2) yes LFKSATTTVMNPKFAES (769) (726) EFAKFEEERARAKWDTANN BETA 3 (3) yes PLYKEATSTFTNITYRGT (762) 20 (1028) RSLPYGTAMEKAQLKPPAT ALPHA 5 (5) no SDA (1049) 1. Argraves et al., 1987, J.C.B. 105:1183-1190 2. Kishimoto et al., 1987, Cell 48:681-690 25 3. Fitzgerald et al., 1987, J.B.C. 262:3936-3939.

EXAMPTT~ VIII
Ca BINDING ASSAY
::
Binding of 4~Ca2 to Fibulin was performed according to the method of Maruyama et al. J. Biochem. 95:511-519 (1984), which is incorporated herein by reference. Briefly, affinity chromatography purified Fibulin was separated by SDS-PAGE on 7.5% acrylamide gels under reducing conditions and then electrophoretically transferred to nitrocellulose filter paper (Towbin et al., Proc. Natl Acad. Sci USA, 76:4350 (1979), which is incorporated herein by reference).
The protein transfers were washed in 60 mM KCl, 10 mM
imidazole-HCl pH 6.8, 0.1% BSA. 45Ca2 (NEN, Boston, MA) at 1 ~Ci/ml in the same buffer was lncubated with the filters for 20 minutes at room temperature. Unbound 45Ca2+ was removed by washing the filters twice for 5 minutes with 50%

2~8~8 W~91/02755 . PCT/US90/0466 ethanol. The filters were then air dried and used to expose Kodak XAR-5 film for 18 hours at -70C.

EXAMPLE IX
INDIRECT IMMUNOFLUORESCENT MICROSCOPY
WITH FIBULIN AND FIBRONECTIN RECEPTOR ANTIBODIES

Cells were seeded onto fibronectin (25 ~g/ml) coated fluorescent microscopy slides (Carlson Scientific, Inc., 10 Peotone, IL) at 1. 6 x 104 cells/ml and grown for 2-8 hours as described in Argraves, et al., Cell 58:623-629 (1989), which ls incorporated herein by reference. The cells were fixed for 30 minutes with freshly prepared 3. 7%
paraformaldehyde (Fluka, Buchs, Switzerland), 0.1% Triton X-100 in PBS. The slides were washed with PBS and then incubated in 3% normal goat serum in PBS (PBS-serum and incubated with the slides for 2 hours at 37C. Rabbit anti-100 kd protein was used at a dilution of 1: 500. In immunoblot analysis this antiserum showed no detectable reactivity for fibronectin receptor. Mouse monoclonal anti-fibronectin receptor B subunit IgG (designated 442) was used at a concentration of 150 ~g/ml. Following incubation with the primary antibodies, the slides were washed with PBS 3 times (5 minutes each wash). The fluorochrome conjugated antiserum (fluorescein goat anti-rabbit IgG, or rhodamine goat anti-mouse IgG; Cappel, West Chester, PA) was diluted (1:40) in PBS-serum and incubated with the slides for 20 minutes at room temperature. The slides were again washed 3 times with PBS. A solution of 40% glycerol in PBS was applied to the upper surfaces of the slides. A glass cover slip was overlaid onto the surface of the glycerol solution and the edges of the cover slip sealed with clear nail polish. In those experiments in which non-permeabilized cells were examined, cells were treated as described above except that Triton X-100 was omitted from the fixation solution.

~ ' , ' .'", : .

wos1/o2755 PCT/US90/04662 -2~6~1 8 32 Stained cells were examined and photographed using an Olympus BHS microscope equipped for fluorescent microscopy.
Photographs were taken using Fujichrome 1600 reversal film shot at ASA 400.

EXAMPLE X
IMMUNOFLUORESCENT LOCALIZATION OF FIBULIN, FIBRONECTIN RECEPTOR AND FIBRONECTIN

This example shows an independent immunofluorescent experiment than that of Example IX which extended staining periods beyond 6 hours after platin~.

Human gingival fibroblasts were seeded at a density of 1.5 X 104 cells/ml onto Lab-Tek chamber slides (Nunc Inc., Naperville, IL) coated with bovine fibronectin (10 ug/ml, Telios Pharmaceuticals, La Jolla, CA). Cells were fixed .
for 30 minutes with 3.7~ paraformaldehyde (Fluka, Buchs, Switzerland), 0.1% Triton X-100 in phosphate-buffered-saline pH 7.2 ~PBS). In indicated experiments, the detergent was omitted from the fixing solution. The slides were washed with PBS and then incubated in 3% normal goat serum, PBS (PBS-serum) for 1 hour at room temperature. The primary antisera were diluted in PBS-serum and incubated with the fixed cells for 2 hours at 37~C. The slides were then washed with PBS three times 5 minutes. The fluorochrome-con~ugated antisera, either fluorescein conjugated sheep anti-mouse IgG or rhodamine conjugated goat anti-rabbit IgG (Cappel, West Chester, PA) were diluted 1:40 in PBS-serum, and incubated with the slides for 20 minutes at room temperature. The slides were again washed with PBS. A solution of 50% glycerol in PBS was applied to the surface of the slides and a glass coverslip overlaid and fixed to the surface with clear nail polish.

Stained cells were examined and photographed using an Olympus BHS microscope equipped for fluorescent microscopy -:- " .' . - ' ' ~'~.' ' '' '.' . ' .
.. . . , . - ,- . :

W091/02755 2 0 6 ~ 8 1 ~ PCT/US90,04662 and having additional exciter filters so as to narrow wavelength bands and restrain crossover excitation during double-fluorochrome label experiments. Photographs were taken using Fujichrome 1600 reversal film with exposure settings controlled with an Olympus model PM-lOADS exposure control unit.

The results showed that Fibulin and the integrin B1 subunit, at early time periods again appeared in immunofluorescent staining as numerous colocalizing streak-like accumulations. In the absence of permeabilizing agent, such staining patterns were not evident. At periods beyond 6 hours the immunofluorescent staining pattern showed that Fibulin accumulated into extensive fibrillar patterns. Furthermore, the fibrillar staining pattern was apparent in the absence of permeabilizing agent which indicated that the immunologically detected Fibulin was extracellular.
.
It was also apparent that the meshwork staining pattern of Fibulin was similar to that of fibronectin.
When double-label immunofluorescent staining was done using antibodies to both Fibulin and fibronectin, very similar staining patterns were seen both at the early and late periods of culture. The staining patterns obtained using Fibulin antibodies could be completely blocked by pre-incubation of the antibodies with 25 ug/ml Fibulin. In addition, pre-incubation of Fibulin antibodies with human fibronectin at 25 ug/ml failed to block antibody staining of Fibulin. These results indicated that Fibulin, like fibronectin, accumulates extracellularly, forming dense networks of fibrils.

.

~ .
"'. ' " ~,: . - .

: ~ ; :

WO91/0275~ 2 ~ ~ 4 ~ ~ ~ 34 PCT/US90/04662 EXAMPLE XI
BIOTINYLATION OF FIBULIN

To evaluate the ability of exogenously added Fibulin to bind to cell monolayers, and become incorporated into a matrix, biotinylated Fibulin was incubated with fi~roblast monolayers. In parallel experiments, biotinylated fibronectin and human IgG were also incubated with fibroblast monolayers as control proteins. After 12 hours of incubation, the exogenously added biotinylated Fibulin was found bound to the cell monolayer, accumulating in elaborate fibrillar networks. A similar pattern of incorporation was obtained with biotinylated fibronectin, but not with biotinylated IgG. The patterns of incorporation of exogenously added Fibulin and fibronectin closely resembled the patterns of endogenous matrix accumulation for each protein as described in Example X.

The Fibulin used for biotinylation was purified by immunoadsorbtion from extracts of human placenta. Ground placental tissue was extracted with 4 M KSCN. Extracts were then clarified by centrifugation, dialyzed against TBS, 10 mM EDTA and passed over a column of plain Sepharose CL-4B. The flow through was then applied to an affinity matrix of monoclonal 5D12/H7 IgG coupled to Sepharose. The column was washed with 0.5 M NaCl, 50 mM Tris pH 7.4, and bound Fibulin eluted with a solution of 4 M KSCN. The eluted Fibulin was dialyzed against TBS and affinity selected on WGA-agarose (see below). Purified Fibulin was incubated with sulfo-N-hyroxysuccinimide-biotin (S-NHS-biotin, Pierce, Rockford, IL) in 0.1 M sodium carbonate pH
8.5 (at a 1:200 molar ratio of protein to S-NHS biotin) for 3 hours at 4C. Following the reaction, the samples were dialyzed against serum-free DMEM supplemented with penicillin, streptomycin, glutamine, sodium bicarbonate, and sodium pyruvate.

wng1/027~s ~ 8 ~ 8 PCT/US90/04662 Gingival cells were grown for 24 hours in Lab-Tek chamber slides coated with 25 ug/ml bovine fibronectin.
Medium was removed and the cell monolayers washed 3 times with serum-free DMEM. Biotinylated-Fibulin, -human fibronectin and -human IgG, each diluted to 0.5 mg/ml in DMEM, ITS (insulin, transferrin, selenous acid, BSA, linoleic acid, Collaborative Research, Bedford, MA) were added separately to the cells and allowed to incubate for 12 hours at 37C. The media were removed and the cell layers washed 3 times with PBS. The fluorochrome conjugate, FITC-avidin (Pierce), was diluted to 30 ug/ml in PBS, was added and incubated for 30 minutes at room temperature. The cell layers were washed 3 times ~ith PBS, mounted and examined by immunofluorescent microscopy as described in Example X.

EXAMPLE XII
INMUNOPRECIPITATION ANALYSIS
"' : .
This example demonstrates the presence of Fibulin in the culture medium which is secreted from fibroblasts.

Nearly confluent human gingival fibroblasts, in 100-mm diameter culture dishes (Becton Dickinson, Lincoln Park, NJ), were radiolabeled for 18 hours with 250 uCi of [35S]-cysteine (New England Nuclear, Boston, MA) in DMEM
(Mediatech, Herdon, VA) containing 10~ bovine calf serum supplemented with iron (HyClone, Logan, Utah). The media was removed and centrifuged at 5000 X g for 15 minutes.
The media supernatant was then dialyzed against 0.5 M NaCl, 2 mM phenylmethysulfonyl fluoride (PMSF), 0.1% Triton X-- 100, 0.1% Tween-20, 50 mM Tris-HCl pH 7.4 (wash buffer) for 18 hours at 4C. The dialyzed media was pre-cleared with 1/50 th volume of protein A-Sepharose (Sigma, St. Louis, MO, mixed 1:1 v/v in wash buffer). Following a 1 hour incubation, the protein A-Sepharose was removed by '.~

; - . ~ . , : .

, '' ..,. ,:
,, : .

WO91/02755 ~ 8 ~ 8 PCT/US90/~4662 -.

centrifugation at 2500 X g for 5 minutes. Antiserum (2 ul) was added to 2 ml aliquots of media and incubated for 18 hours at 4C. Immune complexes were precipitated with protein A-Sepharose and washed repeatedly in wash buffer.
After a final wash in Tris-buffered-saline pH 7.4 (TBS), bound protein was released by addition of SDS
electrophoresis sample buffer and analyzed by SDS-PAGE on 7.5% gels.

The results indicated that Fibulin antibodies immunoprecipitated a single polypeptide with an apparent reduced molecular weight of 100 ~d which corresponded to Fibulin. In the absence of reducing agent, the immunoprecipitated polypeptide exhibited an increased electrophoretic mobility characteristic of Fibulin. The results indicated that Fibulin is secreted by the cultured fibroblasts.

EXAMPLE XIII
PULSE-CHASE IMMUNOPRECIPITATION ANALYSIS
~:, To examine the temporal biosynthesis of Fibulin, pulse-chase immunoprecipitation analyses was performed.
Human gingival fibroblasts were grown to near confluence in 35-mm diameter culture dishes. Cell layers were washed three times with cysteine-free RPMI-1640 (Gibco Laboratories, Grand Island, NY) supplemented with ITS and 10 mM Hepes pH 7Ø The cells were then grown for 15 minutes at 37~C in the cysteine-free RPMI, ITS medium. The cultures were then pulse-labeled for two minutes with cysteine-free RPMI, ITS medium containing 0.5 mCi/ml [35S ] -cysteine. After the 2 minute pulse labeling the medium was removed and the cell layers washed three times with RPMI, ITS containing 1 mM unlabeled cysteine, 10 mM Hepes pH 7.0 and then allowed to incubate for various periods of time in the same medium at 37~C. At the appropriate time intervals, medium was isolated and the cell layer extracted :...... . .: -VV~ 91/02755 2 0 ~ ~ 81 8 PC~r/US90/04662 with 1 ml of 1% Triton X-loo, 0.5 M NaCl, 0.05% Tween 20, 0.05 M Tris-HCl pH 7.4, 2 mM PMSF using a disposable cell scraper. The cell extracts and culture medium fractions were clarified by centrifugation at 100 K X g in a Bec~man model TL-100 ultracentrifuge. The resulting supernatants were pre-absorbed with protein A-Sepharose, used in immunoprecipitation and analyzed by SDS-PAGE as described above. Following SDS-PAGE, gels were treated with Enlightning (NEN Research Products, Boston, MA), dried, and lo used to expose X-ray film at -70C.

The results indicated that within the first minutes of chase two immunoreactive polypeptides of approximate 80 and loo kd molecular weight were present in the cell layer extract. After 5 minutes of chase the level of 80 kd polypeptide diminished. Between 30 and 60 minutes of chase, 100 kd Fibulin polypeptide appeared in the medium with a subsequent decrease in the 100 kd polypeptide in the cell extracts. The results demonstrate a precursor-product relationship between the 80 and 100 kd polypeptides. The 80 kd band may then correspond to the nascent Fibulin polypeptide which is subsequently processed to the 100 kd molecule that is secreted.

ELISA FOR DETERMINING FIBULIN
CONCENTRATION IN PLASMA

To determine the amount of Fibulin in plasma, a two-antibody sandwich ELISA was developed. Microtiter wells were coated overnight with 3 ug/ml mouse anti-Fibulin monoclonal 5D12/H7 IgG in 0.1 M sodium carbonate buffer, pH
9.5. Non-specific binding sites were quenched by addition of 1 mg/ml BSA in PBS. Human plasma, pooled from 5 donors, was serially diluted and incubated with the antibody coating for 1 hour at room temperature. Rabbit anti-Fibulin serum at a dilution of 1:10,000 was incubated for ~ -, ~ , , .:
, ~.

WO91/02755 ~8~8 38 PCT/US90/04662 -1 hour at room temperature followed by goat anti-rabbit IgG
alkaline phosphatase for an additional hour. The chromogenic substrate p-nitrophenyl phosphate (Sigma Chemical Co., St. Louis, MO) was used to measure enzymatic activity bound to the wells. Resulting absorbance values of the plasma samples were compared to those of a serially -diluted standard of purified placental Fibulin. The concentration of the Fibulin standard was determined by protein-dye binding assay (Bradford, Analytical Biochemistry, 72:248-254, 1976, which is incorporated herein by reference). The amount of Fibulin in plasma was determined to be 33 + 3 (mean, + S.D.) ug/ml and SDS-PAGE
analysis showed that the immunologically selected polypeptide displayed electrophoretic properties 15 indistinguishable from Fibulin. ~

EXAMPLE X~ - -LECTIN AFFINITY CHROMATOGRAPHY OF FIBULIN
:' ' .
Fibulin was purified from placental extracts by affinity chromatography on the synthetic B1 subunit cytoplasmic domain peptide-Sepharose as previously described in Example I. Fibulin, in 25 mM octyl-~-D-glucoside, 20 mM EDTA, 2 mM PMSF, TBS was then applied to a column of wheat germ agglutinin (WGA) coupled to agarose (Vector Laboratories, Burlingame, CA) equilibrated in the same buffer. The column was washed with 10 column volumes of TBS and eluted with 2 column volumes of TBS containing, 0.5 M N-acetyl-D-glucosamine (Sigma). Eluted protein was electrophoresed on SDS-polyacrylamide gels and protein bands stained using Coomassie blue.
, , Chromatography of placental Fibulin preparations on columns of WGA coupled to agarose and subsequent SDS-PAGE
analysis revealed that Fibulin bound to the lectin and could be eluted using a solution of the sugar N-acetyl-; glucosamine. No 100 kd polypeptide was found in the .. .

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W~91/027~5 2 ~ 6 4 8 1 ~ PCT/~S90,04662 material that passed through the lectin column which indicated that virtually all the Fibulin had bound. These results indicated that Fibulin is a glycoprotein containing N-acetyl-glucosaminyl carbohydrate constituents.

EXAMPLE XVI
DETECTION OF N-LINKED OLIGOSACCHARIDES

To determine the presence of N-linked oligosaccharides on Fibulin, WGA-agarose selected Fibulin as described in Example XV was first boiled for 3 minutes in 0.5% SDS, 0.1 M ~-mercaptoethanol and then digested with N-glycosidase F
(Genzyme, Boston, MA), according to the manufacturer's protocol, for 18 hours at 37C. Following the digestion, samples were analyzed by SDS-PAGE.

The electrophoretic mobility of Fibulin increased after digestion with the N-glycosidase F. The mobility of the digested material corresponded to a molecular weight of 95 kd. Controls in which Fibulin preparations were incubated under similar conditions, without the enzyme, showed no change in electrophoretic mobility. Assuming a molecular weight of 1500 for an average N-linked carbohydrate-side chain, native Fibulin may then have three N-linked oligosaccharide chains.
.
EXAMPLE XVII
RNA HYBRIDIZATION ANALYSIS

RNA hybridization analysis was performed using a Fibulin cDNA fragment common to the three types of cDNA
(bases 84-234, (Figure 3) as a probe. Briefly, human placental poly(A) RNA was electrophoresed in denaturing 0.8~ agarose gels containing 6% formaldehyde (Lehrach et al., Biochemistry 16:4743-4751, 1977, which is incorporated herein by reference) and blot transferred to nitrocellulose (Thomas, Proc. Natl. Acad. Sci., USA, 77:5201-5205, 1980, .
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WO91/02755 ~ ~ 6 ~ PCT/US90/04662 which is incorporated herein by reference). The filters were probed with a 150 bp DNA segment generated by polymerase chain reaction (PCR) (Saiki et al., Science, 239:487-491, 1988, which is incorporated herein by reference) using a Fibulin cDNA insert as template and upstream and downstream oligonucleotide primers both taken from a region of Fibulin cDNA common to the three cDNA
types. After hybridization the filters were washed under high stringency and used to expose X-ray film at -70C.

The results obtained showed two transcripts of approximately 2.4 and 2.7 kb to be present in human placental poly(A) RNA.

EXAMPLE XVIII
POLYMERASE CHAIN REACTION ANALYSIS

To verify that all the isolated cDNAs corresponded to actual transcripts expressed in placental tissue, a reverse transcriptase polymerase chain reaction (PCR) analysis was performed (Rappolee et al., Science, 241:708-712, 1988, which is incorporated herein by reference). Pairs of synthetic oligonucleotide primers, based on sequence from either side of the divergence point from each cDNA type, were used in PCR to ampli~y cDNA prepared from placental 2S RNA.
, . .
Total human placental RNA (l ug) was used with random hexanucleotide primer (200 ng, Pharmacia), RNasin (30 units, Promega, Madison WI), 1 mM deoxynucleotide triphosphates (dNTPs) and Moloney murine Leukemia virus reverse transcriptase (200 units, Bethesda Research Laboratories, Gaithersburg, MD) to synthesize cDNA. Using one hundredth of the cDNA product, Taq DNA polymerase (3 units, Stratagene, La Jolla, CA), upstream and downstream synthetic oligonucleotide primers (800 ng each), and dNTPs (0.25 mM each) polymerase chain amplification was .. :

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~91/02755 2 0 ~ 4 818 PCT/US90/04662 performed. Primer pairs specific for the cDNA of Fibulin A, B or c, were taken from the following positions within the respective target DNA sequence; 1657-1674 and 2142-2159 for A; 1657-1674 and 2248-2265, for B and; 1442-1459 and 1966-1983 for C. The following temperature parameters were cycled 35 times; 1 minute at 94~C, 2 minutes at the Tm-4C
of the primer with the lower Tm of the given pair, and 3 minutes at 72C. Aliquots of the reactions were analyzed by agarose gel electrophoresis and the separated DNA
stained with ethidium bromide.

The expected sizes for amplified products were 502, 606 and 541 bp for cDNA types A, B and C respectively.
When the products were analyzed by agarose gel electrophoresis, fragments of the appropriate size were obtained, confirming the presence of each transcript in total placental RNA. The product of PCR using the type A
specific primers was repeatedly the lowest in yield. These results indicated that at least three forms of Fibulin transcripts existed, most likely through a process of alternative splicing of a pre-mRNA transcript.

Although the invention has been described in terms of the presently preferred embodiments, it will be apparent to one skilled in the art that modifications can be made without departing from the spirit of the invention. Thus, the invention is limited only by the following claims.

, .

Claims (22)

WE CLAIM:

1. Substantially purified Fibulin.

2. The substantially purified Fibulin of claim 1 having an apparent molecular weight under reducing conditions of 100 kD and which binds to the cytoplasmic domain of the .beta.1 subunit of integrin adhesion receptors in a cation dependent, EDTA reversible manner.

3. A substantially purified polypeptide having substantially the partial amino terminal sequence:
D-V-L-L-E-A-C-C-A-D-G-H-R-M-A

4. A polypeptide having substantially the amino acid sequence encoded by the nucleic acid sequence of Figures 3, 4 or 5.

5. Antibodies reactive with Fibulin or antigenic determinants thereof.

6. The antibodies of claim 5 wherein said antibodies are monoclonal.

7. The antibodies of claim 5 wherein said antibodies are polyclonal.

8. An isolated nucleic acid which encodes or is complementary to nucleic acid encoding Fibulin.

9. The isolated nucleic acid of claim 8, wherein the nucleic acid encoding Fibulin has substantially the sequence as that shown for base pairs 1 through 1707 in Figures 3, 4 or 5.

10. The nucleic acid of claim 9, wherein the nucleotide sequence further comprises substantially the nucleotide sequence as that shown for the base pairs beginning at 1708 and extending to the 3' terminus in Figures 3, 4 or 5.

11. The nucleic acid of claim 8, wherein said nucleic acid is cDNA.

12. A recombinant DNA cloning vector operatively harboring a DNA sequence encoding for Fibulin.

13. A host transformed by the cloning vector of claim 12.

14. A recombinant DNA sequence effective, in compatible host cells, of effecting the expression of Fibulin DNA.

15. A process comprising expressing DNA encoding Fibulin in a host cell.

16. A method for purifying Fibulin from a Fibulin-containing material comprising the steps of:
a. immobilizing a peptide substantially comprising the cytoplasmic domain of .beta.1 integrin subunit on a solid support;
b. contacting said Fibulin-containing material with said immobilized cytoplasmic domain of .beta.1 integrin;
c. removing material not bound to said immobilized cytoplasmic domain of the .beta.1 integrin subunit;
and d. recovering material bound to said immobilized cytoplasmic domain, wherein said recovered material is substantially purified Fibulin.

17. A method of characterizing the extracellular matrix as to the presence of Fibulin, comprising the steps of:

a. contacting said extracellular matrix with antibodies reactive with Fibulin or antigenic determinants thereof; and b. determining whether said antibodies bound to said extracellular matrix.

18. Substantially purified Fibulin having biotin attached thereto.

19. A method of targeting a moiety to the extracellular matrix of an organism comprising a. attaching said moiety to Fibulin, to form a Fibulin complex; and b. administering said Fibulin complex to said organism.

20. The method of claim 19 wherein said moiety is biotin.

21. A method of purifying Fibulin from a Fibulin-containing material comprising the steps of:
a. immobilizing a lectin on a solid support;
b. contacting said Fibulin-containing material with said immobilized lectin;
c. removing material not bound to said immobilized lectin; and d. recovering material bound to said immobilized lectin, wherein said recovered material is substantially purified Fibulin.

22. The method of claim 21, wherein said lectin is wheat germ agglutinin.