WO1993010215A1

WO1993010215A1 - Purified proteoglycan betaglycan, compositions, and methods

Info

Publication number: WO1993010215A1
Application number: PCT/US1992/009956
Authority: WO
Inventors: Joan MASSAGUÉ
Original assignee: Memorial Sloan-Kettering Cancer Center
Priority date: 1991-11-15
Filing date: 1992-11-16
Publication date: 1993-05-27
Also published as: AU3178893A

Abstract

This invention provides a purified betaglycan protein. This invention also provides a purified, soluble betaglycan protein.

Description

PURIFIED PROTEOGLYCAN BETAGLYCAN, COMPOSITIONS, AND METHODS

The invention described herein was made in the course o work under grant No. CA 34610 from the National Institute of Health. The United States government has certain right in this invention.

10

Background of the Invention

Throughout this application, various publications ar referred to within parenthesis. Full bibliographi

15 citations for these references may be found at the end o the specification, immediately preceding the claims. Th disclosures for these publications in their entireties ar hereby incorporated by reference into this application i order to more fully describe the state of the art to which

20 this invention pertains.

Transforming growth factor-β (TGF-β) belongs to a family of growth and differentiation factors that includes the activins and inhibinε, the bone morphogenetic proteins and

25 the Mϋllerian inhibiting substance in mammals, as well as other members identified thus far only in amphibians or insects (Massague, 1990; Roberts and Sporn, 1990) . TGF-β, which exists in various isoforms that are encoded by closely related genes, can inhibit or stimulate cell proliferation,

30 promote extracellular matrix formation, regulate cell differentiation and affect other cellular functions. These responses are observed in cells from virtually every lineage

'<^*• and their intensity may vary with each TGF-β isoform. The mechanism that mediates these responses is unknown. Cell interaction with TGF-β is mediated by a complex grou of proteins that include two high affinity receptors (K 5-30 x 10^~12 M) (receptors I and II) with presumed signa transducing activity (Cheifetz et al., 1987; Boyd an Massague, 1989; Laiho et al. , 1990), the membrane-anchore proteoglycan betaglycan (formerly, the type III receptor) (K_D = 3-30 x 10"¹¹) (Massague and Like, 1985; Cheifetz e al., 1987; Cheifetz et al. , 1988a; Cheifetz et al. 1988b; Segarini and Seyedin, 1988; Cheifetz et al., 1990), and various lower affinity receptors (Cheifetz et al., 1988c; MacKay and Danielpour, 1991; O'Grady et al. , 1991; Cheifetz and Massague, 1991) , some of which are glycolipid-anchored binding proteins specific for different TGF-β isoforms (Cheifetz and Massague, 1991) . The phenotype of certain TGF-β-resis ant cell mutants suggests that initiation of multiple TGF-β responses involves the high affinity receptor types I (53 kda) and II (73 kDa) (Boyd and Massague, 1989; Laiho et al., 1991).

The TGF-β receptors are low-abundance proteins which can be present at levels as low as 100 copies per cell (Boyd and Massague, 1989) . Betaglycan is the most abundant of the three receptors, reaching a level of over 10⁴ copies per cell in some cell lines (Cheifetz et al., 1987; Cheifetz et al. , 1988a; Cheifetz et al. , 1988b; Cheifetz et al., 1988c). The primary structure of these proteins and the nature of their signaling mechanism have not yet been described. However, the recently deduced structure of a receptor for a related factor, activin, suggests that this protein could be a ligand-activated serine/threonine protein kinase (Mathews and Vale, 1991) . This raises the possibility that a new class of receptor-coupled kinases might play a central role in signal transduction by activin and other members of the TGF-β family. * Betaglycan affinity-labelled with radiolabelled TGF- ,y migrates on SDS-polyacrylamide gels as a diffuse band in th

* region of 200-400 kDa. In most cells types examined, betaglycan carries heparan sulfate as well as chondroiti

5 sulfate glycosaminoglycan (GAG) chains. Treatment of th proteoglycans with the GAG-removing enzymes heparitinase an chondroitinase ABC combined generates a core protein of 110- 130 kDa (Cheifetz et al., 1988b; Segarini and Seyedin, 1988) that contains approximately 10 kDa of N-linked carbohydrate

10 (Cheifetz et al., 1988b). Binding of TGF-β is to this core protein (Cheifetz and Massague, 1989; Andres et al., 1991). Studies with cell mutants defective in GAG synthesis have shown that the GAG chains are not required for binding of TGF-β to betaglycan and appear to have no effect on the

15 level of betaglycan at the cell surface or on its affinity for TGF-βl (Saunders et al., 1989). In addition, betaglycan can bind basic fibroblast growth factor (bFGF) via its heparan sulfate chains, and the composition of these chains can be regulated in response to bFGF.

20

In addition to its existence as a membrane component, betaglycan exists in soluble forms that are released by cells into the medium and are found in extracellular matrices and serum (Andres et al., 1989). Betaglycan also

25 occurs naturally in the nonproteoglycan form, particularly in fibroblasts (Cheifetz et al., 1988b).

Betaglycan is present in mesenchymal, epithelial, neuronal and other cell types (Massague et al., 1990) , and seems more 30 abundant in fetal tissues than in adult tissues, suggesting a role in development (Andres et al., 1991). However, betaglycan is not expressed in certain types of myoblasts, endothelial, epithelial and hematopoietic cells which nevertheless respond to TGF-β (Massague et al., 1986; Ohta et al., 1987; Segarini et al., 1989; Cheifetz et al., 1990). Betaglycan is retained in each one of a large panel of mutant cell clones that are resistant to TGF-β due to loss of receptors I or II (Boyd and Massague, 1989) . These observations suggest that betaglycan may not be directly involved in transmembrane signalling despite the existence of a correlation between the relative affinity of various TGF-β isoforms for betaglycan and their biological potency in some cell lines (Cheifetz et al. , 1987). Rather than being directly involved in signal transduction, betaglycan might act to control the access of TGF-β to the signaling receptors. The widespread distribution of betaglycan in avian and mammalian cells and its high affinity for TGF-β suggest that betaglycan may play important roles possibly as a regulator of the availability of bioactive TGF-βs to signalling receptors while the GAG chains might be involved in interactions with other growth factors and/or the extracellular matrix (Segarini and Seyedin, 1988; Andres et al., 1989; Cheifetz and Massague, 1989).

The importance of such growth factor receptor accessory molecules has been recently demonstrated in the FGF system. bFGF binding to the heparan sulfate chains of certain membrane proteoglycans is required for its binding to signaling receptors (Yayon et al., 1991; Rapraeger et al.,

1991) . These are receptors that mediate effects of FGF on proliferation and differentiation of mesenchymal and neuroectodermal cells (Lee et al. , 1985; Folkman and

Klagsbrun, 1987) . As a member of the heparin-binding growth factor family, bFGF binds to the heparan sulfate glycosaminoglycan (GAG) chains of pericellular proteoglycans

(Moscatelli, 1988) one of which is syndecan (Saunders et al., 1989; Kiefer et al. , 1990) . Binding of bFGF to heparan sulfate is required before bFGF binds to the signalling <_. receptor, as can be shown with cell mutants defective in GA

_Λ synthesis (Yayon et al., 1991) or treatment of cells wit heparitinase (Rapraeger, et al., 1991). Other GAGs such a chondroitin sulfate cannot substitute for heparan sulfate i

5 this role (Yayon et al., 1991).

Betaglycan is thus an example of an emerging class o low-abundance membrane-bound proteoglycans whose role is t mediate cell interaction with or recognition o 10 extracellular molecules that control cell proliferation differentiation and organization (Ruoslahti and Yamaguchi 1991; Kjellen and Lindahl, 1991).

Sum ary of the Invention

This invention provides an isolated, purified betaglyca protein. This invention also provides a purified, solubl betaglycan protein.

Brief Description of the Figures

Figure l, DEAE-trisacryl chromatography of betaglycan Solubilized fetal rat membranes (3 membrane protein from 0.5 kg tissue) wer chro atographed over a 250-ml DEAE-trisacry column at pH 6.0 and eluted with a 300 mM 1.5 M NaCl gradient. A: Elution profile o betaglycan. The solid line corresponds t the absorbance of the eluate at 280 nm (ful scale = 2.0 absorbance units). Square represent the salt concentration. The pea of betaglycan elution was determined b affinity labelling of the eluting fractions electrophoresis through polyacrylamide gels and excision and quantitation by gamm counting of the labelled bands (triangles) B: Autoradiography of affinity-labelle fractions.

Figure 2, Characteristics of cell-surface and tissue derived betaglycan. A: Affinity labellin competition assay of betaglycan on Rat- cells and in solubilized membranes from ra fetal tissue. Samples were affinit labelled with 50 pM ¹²⁵I-TGF-B1 in th presence of increasing concentrations o unlabelled TGF-βl or TGF-β2. Rat 1 cell are shown in the left panel and solubilize fetal rat membranes at right. The type (R-I) and type II (R-II) receptors as wel as betaglycan (BG) are indicated. B Enzymatic deglycosylation of affinit labelled betaglycan. Detergent extract from Rat 1 cells (left) or solubilized feta rat membranes (right) were affinity labelle with 150 pM ¹²⁵I-TGF-B1 and subjected t deglycosylation by heparitinase (H) , chondroitinase ABC (C) , or a mixture of th two enzymes (H/C) . The position of betaglycan and of the deglycosylated core are indicated.

Figure 3 TGF-β agarose affinity chromatography of betaglycan. The gradient was as indicated in the text, and spanned fractions 1 to 20. A: Affinity labelling of fractions eluted with a pH and salt gradient. B: Bolton- Hunter labelling of the eluted fractions.

Figure 4. Characterization of purified betaglycan. A: Affinity labelling and enzymatic digestion of purified betaglycan. Purified betaglycan was affinity labelled with 150 pM ¹²⁵I-TGF-B1 and subjected to enzymatic digestion by heparitinase (H) , chondroitinase ABC (C) , or a mixture of the two enzymes (H/C) . B: Enzymatic digestion of Bolton-Hunter labelled betaglycan. C: The band obtained by digestion of ¹²⁵I-Bolton-Hunter-labelled betaglycan was excised from the gel and treated with (F) or without (0) endoglycosidase F.

Figure 5. Affinity labelling competition assay of purified betaglycan. Purified betaglycan was affinity labelled with 50 pM ¹²⁵I-TGF-βl in the presence of increasing concentration of unlabelled TGF-βl or TGF-B2 as shown.

Figure 6, Liposo e association of purified betaglycan Phosphatidylcholine liposomes were formed i the presence of affinity labelled purifie betaglycan and separated from unincorporate material on a sucrose gradient. A Distribution of betaglycan. B: Distributio of the liposome marker, ¹⁴ C phosphatidylcholine. Fractions are numbere starting from the top of the gradient.

Figure 7 Purification of the core protein o betaglycan. Wheat germ lectin-purifie betaglycan was treated with heparitinase an chondroitinase ABC and chromatographed o TGF-βl agarose. A: Elution of betaglyca core as determined by affinity labellin with ¹²⁵I TGF-βl, electrophoresis throug polyacrylamide gels, and quantitation b phosphorimager. B: ¹²⁵I-Bolton-Hunte labelling of the eluting fractions. C: Association of the core protein wit phosphatidlycholine liposomes. The to panel shows the distribution of betaglyca core. The bottom panel shows th distribution of the liposome marker. Fractions are numbered starting from the to of the gradient.

Figure 8 Betaglycan core protein purification an betaglycan cDNA cloning strategy. A: Activ betaglycan core protein fraction eluted fro TGF-β1-sepharose and labeled with ¹²⁵I-Bolton-Hunter reagent (Andres et al., 1991) , as detected by SDS-PAGE (5-15% linear polyacrylamide gradient) and autoradiography. B: The amino acid sequence of betaglycan tryptic fragment CT90, the synthetic oligonucleotide pools used as primers in the PCR reaction, and the oligonucleotide jd4 synthesized according to the sequence of the product that was amplified in the PCR reaction. C: Scheme of the inserts present in the six positive clones that were isolated from a rat fibroblast lambda gtll library by screening with jd4. The box in clone bg7 indicates the location of the long open reading frame. The approximate location of the jd4 sequence is indicated.

Figure 9 , Nucleotide sequence of the rat betaglycan cDNA clone bg7 and deduced amino acid sequence. The experimentally determined sequences- of betaglycan tryptic fragments are underlined. Peptide CT90 corresponds to residues 629-650 of the predicted protein. The hydrophobic regions in the putative signal sequence and transmembrane region are shown with double overlining. Potential GAG chain attachment sites (o) some of which are in the vicinity of acidic residues (_) , and N-linked glycosylation sites (o) are indicated. The two potential cleavage sites for release of soluble betaglycan are overlined. Figure 10, Structural features of betaglycan co protein predicted from its cDNA. Shown a the location of the hydrophobic N-termin region (black box) , the transmembrane regi (stippled box) , the six potential N-link glycosylation sites (o) , the most favorab (o) and other (o) potential GAG attachme sites, the seventeen cysteines (*) , t Lys-Lys potential cleavage site (KK) , t potentially regulated Leu-Ala-Val-V cleavage site (A/V) , the proline-rich regi (P) , the serine/threonine-rich C-termin region (S/T) and the region of homology endoglin.

Figure 11, Alignment of the C-terminal portion of t rat betaglycan and human endoglin amino ac sequences. Identical amino acids (|) a conservative substitutions (:) a indicated.

Figure 12 Expression of rat betaglycan cDNA. A: COS cells were transfected with pCMV5 vect alone, or this vector containing t betaglycan clone bg7 in the sense (pCMV-b or antisense (pCMV-gb) orientations relati to the CMV promoter. Three days late cells were affinity-labelled with ¹²⁵I-TGF- alone or in the presence of competi TGF-βl. Some cultures were incubated wi chondroitinase ABC (C) , heparitinase (H) both enzymes (C/H) or buffer alone (- before incubation with ¹²⁵I-TGF-βl Affinity-labelled cell extracts we subjected to SDS-PAGE and autoradiography. The positions of the ¹²⁵I-TGF-βl-labelled proteoglycan (BG) and its core product generated by enzyme digestion are indicated. B: COS-1 cells transfected with pCMV-bg were affinity-labelled 3 days later with 50 pM ¹²⁵I-TGF-βl alone (-) or in the presence of 5 nM TGF-βl or TGF-β2. Both TGF-β isoforms inhibited the labelling of betaglycan. C: Fetal bovine endothelial cells which do not express endogenous betaglycan were transfected with pCMV-gb (gb) or pCMV-bg (bg) . Three days later, cells were affinity-labelled with ¹²⁵I-TGF-B1 alone or in the presence of excess TGF-βl. Labelled cell extracts were subjected to SDS-PAGE and autoradiography. FBHE cells expressed the transfected betaglycan as a mixture of proteoglycan form (BG) and core lacking GAG chains (Core) . The positions of the endogenous TGF-β receptors I and II are also indicated.

Figure 13. Expression of the soluble betaglycan form. COS-1 cells, Tl cells, and FBHE cells were transfected with pCMV-gb or pCMV-bg. Media conditioned by these cells during days 2 and 3 after the transfection were collected and chro atographed over DEAE-trisacryl to isolate the proteoglycans. This fraction was then affinity-labelled with ¹²⁵I-TGF-βl and subjected to SDS-PAGE and autoradiography to visualize soluble betaglycan. Figure 14 Northern blot analysis of betaglycan mRNA Poly(A)⁺ RNA (2.5μg) from Rat-1 fibroblasts and mouse 3T3 fibroblasts which expres endogenous betaglycan, and from L₆E₉ ra skeletal myoblasts which do not expres betaglycan, were electrophoresed on denaturing agarose gel and subjected t Northern blot analysis using ³²P-labeled bg cDNA as a probe. An mRNA species o approximately 6 kb was detected in the Rat- and 3T3 cell samples.

Figure 15, Binding of bFGF to betaglycan and it enzymatically generated core protein.

Figure 16, Effect of bFGF on the migration o betaglycan through polyacrylamide gels. F3 rat calvaria osteoblast cultures were grow to confluency and treated with or without 0.6 nM bFGF before affinity labelling cell surface proteins ¹²⁵I-TGF-βl in the presence of increasing concentrations of TGF-βl as indicated. Affinity labelled complexes corresponding to betaglycan (BG) , and TGF-β receptor components I (R-I) and II (R-II) are indicated. Numbers on the left indicate the molecular weight (m.w. ) of protein markers in kDa.

Figure 17 Loss of heparan sulfate in betaglycan from bFGF-treated cells. Osteoblasts treated with or without bFGF were affinity labelled with ¹²⁵I-TGF-βl and digested with heparitinase, chondroitinase ABC or with a mixture of the two enzymes. Extracts from affinity labelled cells were incubated with heparitinase, chondroitinase ABC or both enzymes. BG-C: Betaglycan core protein 5 containing chondroitm sulfate. BG-core:

Betaglycan core protein. BG-HH: Betaglycan protein with a high heparan sulfate concentration. BG-LH: Betaglycan core protein with a very low heparan sulfate 10 concentration.

Detailed Description of the Invention

This invention provides a purified betaglycan protein. Thi molecule and its equivalents were obtained by the mean described below. For the purposes of this invention, "purified betaglycan protein" is a protein free of othe proteins and cellular components.

The purified betaglycan protein is a mammalian-derive protein. For example, the protein may be purified fro murine cells or human cells. In one embodiment of thi invention, the purified betaglycan protein has a molecula weight of from about 200 kDa to about 400 kDa. However, i the preferred embodiment of this invention, the purifie protein has a molecular weight of from about 280 kDa t about 330 kDa. The purified betaglycan protein may have but is not limited to having, the amino acid sequence show in Figure 9, or a fragment thereof.

In one embodiment of this invention, the purified betaglyca protein is capable of binding a molecule of the transformin growth factor TGF-β. In another embodiment of thi invention, the purified betaglycan is capable of binding molecule of basic fibroblast growth factor. However, in th preferred embodiment of this invention, the purifie betaglycan protein is capable of binding a molecule of th transforming growth factor TGF-β and a molecule of basi fibroblast growth factor (bFGF) .

The betaglycan protein described and claimed herein i valuable for the information it provides concerning th nucleotide sequences encoding it. The nucleotide sequence can be used to produce the soluble protein described an claimed herein. The betaglycan protein is also valuable a a product in protein complexes and pharmaceutical compositions valuable in new and useful methods described and claimed herein.

This invention also provides a nucleic acid molecule encoding the betaglycan protein of this invention, e.g., a genomic molecule. This molecule and its equivalents were obtained by means described below. In one embodiment of this invention, the nucleic acid molecule is a DNA molecule. In another embodiment of this invention, the DNA molecule is a cDNA molecule, e.g., a cDNA molecule having a nucleotide sequence substantially the same as the nucleotide sequence shown in Figure 9 or a fragment thereof.

The nucleic acid molecules described and claimed herein are useful for generating new viral and circular plasmid vectors described and claimed herein. The nucleic acid molecules are also valuable in a new and useful method of gene therapy, i.e., by stably transforming cells isolated from an animal with the nucleic acid molecules and then readministering the stably transformed cells to the animal. Methods of isolating cells include any of the standard methods of withdrawing cells from an animal. Suitable isolated cells include, but are not limited to, bone marrow cells. Methods of readministering cells include any of the standard methods of readministering cells to an animal.

This invention also provides a purified, soluble betaglycan protein. This molecule and its equivalents were obtained by the means described below. For the purposes of this invention, a "purified, soluble betaglycan protein" is a betaglycan protein free of cell membranes and other cellular components. The soluble betaglycan protein may have, but is not limited to having, the amino acid sequence of th sequence shown in Figure 9, or any fragment thereof.

In one embodiment of this invention, the soluble betaglyca protein is capable of binding a molecule of the transformin growth factor TGF-β. In another embodiment of thi invention, the soluble betaglycan protein is capable o binding a molecule of basic fibroblast growth factor However, in the preferred embodiment of this invention, th soluble protein is capable of binding a molecule of th transforming growth factor TGF-β and a molecule of basi fibroblast growth factor.

In one embodiment of this invention, the soluble betaglyca protein is labelled with a detectable marker, for example a radioactive isotope, enzyme or dye. However, an "detectable" marker known to those of skill in the art i contemplated by this invention.

The soluble betaglycan protein described and claimed herei is valuable as a product in protein complexes an pharmaceutical compositions. These complexes an compositions are valuable in new and useful methods of determining the ratio of active to inactive TGF-β in th body of an animal; increasing the concentration of free TGF β in the body of an animal; decreasing the concentration o free TGF-β in the body of an animal; imaging TGF-β in th body of an animal; and increasing the concentration of fre fibroblast growth factor in the body of an animal. Th soluble betaglycan protein is also valuable as a produc useful for the synthesis of the glycosaminoglycan-fre soluble betaglycan protein described and claimed herein. This invention provides a nucleic acid molecule encoding the soluble betaglycan protein, e.g, a genomic molecule. This molecule and its equivalents were obtained by the means described below. In one embodiment of this invention, the nucleic acid molecule is a DNA molecule. In another embodiment of this invention, the DNA molecule is a cDNA molecule, e.g., a molecule having a nucleotide sequence substantially the same as the nucleotide sequence encoding the soluble portion of the betaglycan protein shown in Figure 9, or a fragment thereof.

This invention provides a soluble betaglycan protein free of glycosaminoglycan chains. In one embodiment of this invention, the glycosa inoglycan-free soluble betaglycan protein has a molecular weight of from about 80 kDa to about 130 kDa. However, in the preferred embodiment of this invention, the glycosaminoglycan-free soluble betaglycan protein has a molecular weight of from about 90 kDa to about 93 kDa and is capable of binding a molecule of the transforming growth factor TGF-β. In one embodiment of this invention, the glycosaminoglycan free soluble betaglycan protein is labelled with detectable marker, for example a radioactive isotope, enzym or dye. However, any "detectable" marker known to thos skilled in the art is contemplated by this invention.

The soluble betaglycan protein described and claimed herei is valuable as a product in protein complexes an pharmaceutical compositions. These complexes an compositions are valuable in new and useful methods of: determining the ratio of active to inactive TGF-β in the body of an animal; increasing the concentration of free TGF- β in the body of an animal; decreasing the concentration of free TGF-β in the body of an animal; imaging TGF-β in the body of an animal

This invention provides a protein complex comprising the soluble betaglycan protein bound to a molecule of the transforming growth factor TGF-β and to a molecule of basic fibroblast growth factor. This invention also provides a protein complex comprising the soluble betaglycan protein bound to a molecule of the transforming growth factor TGF-β. This invention further provides a protein complex comprising the soluble betaglycan protein bound to a molecule of basic fibroblast growth factor.

This invention provides a protein complex comprising the glycosaminoglycan-free soluble betaglycan protein bound to a molecule of the transforming growth factor TGF-β.

The protein complexes described and claimed herein are valuable as products in new and useful pharmaceutical compositions. Such compositions are valuable in new and useful methods of: determining the ratio of active to inactive TGF-β in the body of an animal; increasing the concentration of free TGF-β in the body of an animal; decreasing the concentration of free TGF-β in the body of an animal; imaging TGF-β in the body of an animal; and increasing the concentration of free fibroblast growth factor in the body of an animal.

This invention provides a pharmaceutical composition comprising the purified, soluble betaglycan protein and a pharmaceutically acceptable carrier. This invention also provides a pharmaceutical composition comprising the glycosaminoglycan-free betaglycan protein and a pharmaceutically acceptable carrier. This invention further provides a pharmaceutical composition comprising any protein complex described hereinabove and a pharmaceutically acceptable carrier. For the purposes of this invention, "pharmaceutically acceptable carriers" means any pharmaceutical composition generally accepted by those skilled in the art. Examples include, but are not limited to, phosphate buffered saline, physiological saline and human serum albumin.

The pharmaceutical compositions described and claimed herein are valuable as products in new and useful methods of: determining the ratio of active to inactive TGF-β in the body of an animal; increasing the concentration of free TGF- β in the body of an animal; decreasing the concentration of free TGF-β in the body of an animal; imaging TGF-β in the body of an animal; and increasing the concentration of free fibroblast growth factor in the body of an animal.

This invention provides a pharmaceutical composition comprising the nucleic acid molecule encoding the betaglycan protein and a pharmaceutically acceptable carrier. This invention also provides a pharmaceutical compositi comprising the nucleic acid molecule encoding the solub betaglycan protein and a pharmaceutically acceptab carrier. For the purposes of this inventio "pharmaceutically acceptable carriers" means a pharmaceutical composition generally accepted by thos skilled in the art. Examples include, but are not limite to, phosphate-buffered saline and Tris-HCl. Th pharmaceutical compositions provided by this invention ar valuable products useful in isolating human genomic DNA o human cDNA encoding the betaglycan protein. Th pharmaceutical compositions may also be useful for gen therapy in humans and other mammals, i.e., by stabl transforming cells isolated from an animal with the nuclei acid molecules and then readministering the stabl transformed cells to the animal. Methods of isolating cell include any of the standard methods of withdrawing cell from an animal. Suitable isolated cells include, but ar not limited to, bone marrow cells. Methods o readministering cells include any of the standard methods o readministering cells to an animal.

This invention provides a monoclonal antibody specificall reactive with the purified betaglycan protein. For th purposes of this invention, a "monoclonal antibody" is a immunologically reactive molecule derived from monospecific B lymphocyte, i.e., a B lymphocyte which make immunologically reactive molecules against a single antigen binding site. Accordingly, the monoclonal antibod specifically recognizes and reproducibly binds to particular arrangement of atoms on the surface of a antigen. For the purposes of this invention, an "antigen is any substance, cell or tissue capable of eliciting a immune response. In one embodiment of this invention, the monoclonal antibody is labelled with a detectable marker, for example, a radioactive isotope, enzyme or dye. However, any detectable marker known to those skilled in the art is contemplated by this invention. The monoclonal antibody may be a murine or a human monoclonal antibody.

The monoclonal antibody described and claimed herein is valuable as a product in a new and usefule pharmaceutical composition. Such a composition is valuable in a new and useful method of imaging TGF-β in the body of an animal.

This invention also provides a hybridoma cell line which produces the monoclonal antibody. For the purposes of this invention, a "hybridoma cell line" is a cell line produced by fusing an antibody-producing B-lymphocyte with a B- lymphocyte tumor cell. The antibody-producing B-lymphocyte may be a murine or human B-lymphocyte. The B-lymphocyte tumor cell may be derived from a murine spleen.

This invention provides a monoclonal antibody specifically reactive with the soluble betaglycan protein. The monoclonal antibody provided by this invention will also be specifically reactive with the glycosaminoglycan-free soluble betaglycan protein. In one embodiment of this invention, the monoclonal antibody is labelled with a detectable marker, for example, a radioactive isotope, enzyme or dye. However, any detectable marker known to those skilled in the art is contemplated by this invention. The monoclonal antibody may be a murine or a human monoclonal antibody. This invention also provides a hybridoma cell line which produces the monoclonal antibody. The monoclonal antibodies described and claimed herein ar valuable as products in pharmaceutical compositions. Suc compositions are valuable in a new and useful method o imaging TGF-β in the body of an animal.

Thus, a pharmaceutical composition which comprises monoclonal antibody described hereinabove and pharmaceutically acceptable carrier is also provided. Fo the purposes of this invention, "pharmaceutically acceptabl carriers" are well known to those skilled in the art Examples include, but are not limited to, phosphate buffere saline or human serum albumin.

This invention provides a gene transfer vector comprising nucleic acid molecule operably linked to a promoter of RN transcription. For the purposes of this invention, a "gen transfer vector" is a vector, for example, a plasmid vecto or a viral vector, capable of transferring DNA into a cell. In one embodiment of this invention, the nucleic aci molecule is a nucleic acid molecule encoding the purifie betaglycan protein. In another embodiment of thi invention, the nucleic acid molecule is a nucleic aci molecule encoding the soluble betaglycan protein.

This invention provides a host vector system for th production of a betaglycan protein which comprises a gen transfer vector in a suitable host. In one embodiment o this invention, the betaglycan protein is the purifie betaglycan protein. In another embodiment of thi invention, the betaglycan protein is the soluble betaglyca protein. In still another embodiment of this invention, th betaglycan protein is the glycosaminoglycan-free betaglyca protein. In one embodiment of this invention, the suitable host cell is a eukaryotic cell, for example, a yeast cell, insect cell or a mammalian cell. For the purposes of this invention, a "suitable host cell" for the production of the glycosaminoglycan-free betaglycan protein is a host cell defective in glycosaminoglycan synthesis. Cells defective in glycosaminoglycan synthesis are well known to those skilled in the art.

The gene transfer vectors described and claimed herein are valuable as products useful for generating stably transformed eukaryotic host cells, and thereby in new and useful methods for the production of protein comprising growing such host cells under conditions suitable for the production of a protein.

This invention provides a method of producing the betaglycan protein which comprises growing the host vector system for the production of the betaglycan protein under conditions permitting production of betaglycan protein and recovering the betaglycan protein so produced. This invention also provides the protein produced by this method.

This invention also provides a method of producing the soluble betaglycan protein which comprises growing the host vector system for the production of the soluble betaglycan protein under conditions permitting production of soluble betaglycan protein and recovering the soluble betaglycan protein so produced. This invention further provides the protein produced by this method.

This invention further provides a method for the production of the glycosaminoglycan-free betaglycan protein which comprises growing the host vector system for the production -25-

of the glycoa inoglycan-free betaglycan protein und conditions permitting production of glycosaminoglycan-fr betaglycan protein and recovering the glycosaminoglycan-fr betaglycan protein so produced. This invention furth provides the protein produced by this method.

This invention provides a method of purifying the betaglyc protein which comprises: solubilizing cell and tiss membranes containing the betaglycan protein; passing sample of said solubilized membrane through an ion-exchan chromatography column; passing the resulting betaglyca enriched eluate fractions through a lectin chromatograp column; passing the resulting betaglycan-enriched elua fractions through a column containing immobilized TGF-β a recovering the betaglycan from the resulting eluate. Th invention further provides the betaglycan protein purifi by this method.

For the purposes of this invention, an "ion-exchan chromatography column" is a chromatography column design to separate proteins on the basis of their charge. For t purposes of this invention, a "lectin chromatography colum is a chromatography column designed to separate proteins the basis of their sugar content. This invention al provides the protein produced by this method.

This invention further provides a method of producing t glycosaminoglycan-free soluble betaglycan protein whi comprises contacting a sample of the soluble betaglyc protein with an amount of an enzyme composition effective cleave the glycosaminoglycan chains from the core of t soluble betaglycan protein. In one embodiment of thi invention, the enzyme composition comprises heparitinas In another embodiment of this invention, the enzy composition comprises chondroitinase. However, in the preferred embodiment of this invention, the enzyme composition comprises heparitinase and chondroitinase. This invention further provides the protein produced by this method.

For the purposes of this invention, an "effective amount" of an enzyme composition is any amount of enzyme composition which is effective to cleave the glycosaminoglycan chains from the betaglycan protein core. Methods of determining an "effective amount" are well known to those skilled in the art and will depend upon a number of factors including, but not limited to: the size of the sample and the concentration of betaglycan protein in the sample.

This invention provides a method of determining the ratio of active TGF-β in an animal to the total amount of TGF-β in the animal comprising: isolating a suitable sample from the body of an animal; contacting the sample with an amount of the pharmaceutical composition comprising the soluble betaglycan protein and a pharmaceutically acceptable carrier, or the glycosaminoglycan-free soluble betaglycan protein and a pharmaceutically acceptable carrier effective to bind all the active TGF-β in the sample, under conditions suitable to the formation of complexes between betaglycan protein and active TGF-β; and determining the amount of complex formed and comparing the amount of complex formed in the sample to the total amount of TGF-β in the sample.

In one embodiment of this invention, the suitable sample is blood. In another embodiment of this invention, the suitable sample is body tissue. In one embodiment of this invention, the animal is a rat. In another embodiment of this invention, the animal is mammal, e.g., a human.

For the purposes of this invention, an "effective amount" o a pharmaceutical composition is any amount of th pharmaceutical composition effective to bind to all th active TGF-β in the sample. Methods of determining a "effective amount" are well known to those skilled in the art and will depend upon a number of factors including, but not limited to: the size of the sample, the total amount of TGF-β in the sample and the concentration of betaglycan protein in the pharmaceutical composition.

This invention provides a method of increasing the concentration of free TGF-β in an animal which comprises administering to the animal an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and: the soluble betaglycan protein; or the glycosaminoglycan-free soluble betaglycan protein; or a protein complex comprising the soluble betaglycan protein bound to a molecule of the transforming growth factor TGF-β and a molecule of basic fibroblast growth factor; or a protein complex comprising the soluble betaglycan protein bound to a molecule of the transforming growth factor TGF-β; or a protein complex comprising the glycosaminoglycan-free soluble protein bound to a molecule of the transforming growth factor TGF-β.

For the purposes of this invention, an "effective amount" of a pharmaceutical composition is any amount of the pharmaceutical composition which is effective to increase the concentration of free TGF-β in the body of an animal. Methods of determining an "effective amount" are well known to those skilled in the art and will depend on a number of factors including, but not limited to: the type of animal involved, the size of the animal's body and the amount by which the concentration of free TGF-β is to be increased.

In one embodiment of this invention, the animal is a rat. In another embodiment of this invention, the animal is a human.

In one embodiment of this invention, a patient suffering from a condition, for example a wound, a detached retina or a broken bone, can be treated by increasing the concentration of free TGF-β in the body of the patient according to the method of this invention.

This invention provides a method of decreasing the concentration of free TGF-β in an animal which comprises administering to the animal an amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the soluble betaglycan protein or the glycosaminoglycan- free soluble betaglycan protein effective to decrease the concentration of free TGF-β in the body of the animal. This method is intended to include pharmaceutical compositions comprising biologically active fragments of the soluble betaglycan or the glycosaminoglycan-free soluble betaglycan protein, i.e., fragments of the protein capable of binding TGF-β. Preferably, these fragments would not be able to deliver TGF-β to its cellular receptors. These fragments are valuable as non-immunogenic molecules with a high affinity for TGF-β.

For the purposes of this invention, an "effective amount" of a pharmaceutical composition is any amount of the pharmaceutical composition which is effective to decrease the concentration of free TGF-β in the body of the animal Methods of determining an "effective amount" are well know to those with skill in the art and will depend on a numbe of factors including, but not limited to: the type of anima involved, the size of the animal's body and the amount b which the concentration of free TGF-β is to be decreased The method of this invention is valuable to counteract th effects of TGF-β, for example, inhibition of cel proliferation, in the body of an animal suffering from disease, e.g., fibrosis of the lung. The soluble betaglyca protein is valuable as a non-immunogenic molecule with high affinity for TFG-β.

This invention provides a method of imaging TGF-β in th body of an animal which comprises: administering to th animal an amount of a pharmaceutical composition comprisin the soluble betaglycan protein or the glycoaminoglycan-fre soluble betaglycan protein and a pharmaceutically acceptabl carrier effective for the binding of the protein to TGF- throughout the body of the animal, under condition permitting the betaglycan protein to bind TGF-β; administering to the animal an effective imaging amount of the pharmaceutical composition comprising a monoclonal antibody specifically reactive with the betaglycan protei or the soluble betaglycan protein and a pharmaceuticall acceptable carrier under conditions permitting the formation of complexes between the monoclonal antibody and betaglycan protein bound to TGF-β; clearing any unbound imaging agent from the body of the animal; and imaging any monoclonal antibody-betaglycan protein complexes found in the animal.

For the purposes of this invention, an "effective amount" of a pharmaceutical composition is any amount of the pharmaceutical composition which is effective to allow for binding of the soluble betaglycan protein or th glycosaminoglycan-free soluble betaglycan protein to TGF- throughout the body of the animal. Methods of determinin an "effective amount" are well known to those skilled in the art and will depend upon an number of factors including, but not limited to: the type of animal involved, the size of the animal, the amount of TGF-β in the body of the animal and the concentration of betaglycan protein in the pharmaceutical composition.

For the purposes of this invention, an "effective imaging amount" is any amount which is effective to image the monoclonal antibody-betaglycan protein complexes in the body of an animal. Methods of determining an "effective imaging amount" are well known to those skilled in the art and will depend upon an number of factors including, but not limited to the type of animal involved and the size of the animal's body.

In one embodiment of this invention, the animal is a human. In another embodiment of this invention, the administration to the animal comprises intravenous injection.

This invention provides a method of increasing the concentration of free basic fibroblast growth factor in the body of an animal, for example a rat or a human, which comprises administering to the animal an amount of a pharmaceutical composition effective to increase the concentration of free basic fibroblast growth factor in the body of the animal. The pharmaceutical composition comprises a pharmaceutically acceptable carrier and a protein complex described hereinabove. In one embodiment of this invention, the protein complex comprises the soluble betaglycan protein bound to a molecule of the transforming growth factor TGF-β and a molecule of basic fibroblas growth factor. In another embodiment of this invention, th protein complex comprises the soluble betaglycan protei bound to a molecule of basic fibroblast growth factor.

For the purposes of this invention, an "effective amount" o a pharmaceutical composition is any amount of th pharmaceutical composition which is effective to increas the concentration of free basic fibroblast growth factor i the body of an animal. Methods of determining an "effectiv amount" are well known to those skilled in the art and wil depend upon a number of factors including, but not limite to: the type of animal involved, the size of the animal' body and the amount by which the concentration of free basi fibroblast growth factor is to be increased. The method o this invention is valuable to promote fibroblas proliferation in the body of an animal by increasing th concentration of basic fibroblast growth factor in the bod of the animal.

Materials and Methods

AFFINITY LABELLING OF BETAGLYCAN. Betaglycan was identified in solubilized membranes and column fractions by affinity labelling with ¹²⁵I-TGF-B1. Porcine TGF-βl (R&D Systems, Minneapolis, MN) was iodinated using chloramine T. Samples were affinity labelled by incubation with 150 pM ¹²⁵I TGF-βl in binding buffer (50 mM NaCl, 10 mM MgCl₂, 5 mM KCl, 25 mM Hepes, pH 7.6), 0.1% Triton X-100, for 2 hours at 4°C. Bound ligand was cross-linked to betaglycan with disuccinidimyl suberate (Cheifetz et al. , 1988b) . Cross-linking was stopped by the addition of electrophoresis sample buffer or, in preparation for subsequent enzyme treatment, Tris-HCl, pH 7.0, to a final concentration of 0.1 M. Betaglycan was visualized by electrophoresis through SDS-polyacrylamide gels and subsequent autoradiography of the fixed gel (Laemmli, 1970) . The labelled band was then excised and the radioactivity quantitated by gamma counting. For competition studies, unlabelled porcine TGF-βl or TGF-B2 was added at the time of addition of ¹²⁵I-labelled ligand.

PURIFICATION OF BETAGLYCAN. All purification steps were carried out at 4°C. Fetal rat carcasses (500 g) were homogenized in 250 mM sucrose, 25 mM Hepes, pH 7.4, and 1 mM EDTA using a Polytron tissue homogenizer. Large particulate matter was removed by centrifugation for 10 minutes at 3000xg. Pellets were resuspended twice in the same buffer and centrifuged under the same conditions. Supernatants were pooled and membranes pelleted by centrifugation for 1 hour at 30,000xg. The pelleted membranes were washed by resuspension in l mM EDTA, 25 mM Hepes,pH 7.4 , and 300 mM NaCl and pelleted again by centrifugation for 20 minutes at 100,000xg. The membranes were then resuspended in this buffer to a final concentration of 10-20 mg/ l and proteins were solubilized by the addition of 2% Lubrol PX (v/v) a stirring for l hour. Insoluble material was removed centrifugation for 30 minutes at l00,000xg. Supernatan were then loaded onto a 250-ml DEAE-Trisacryl (IBF) radi column (Sepragen, Inc.) equilibrated in 300 mM NaCl, 0. Lubrol PX, 10% glycerol, and 10 mM bisTris, pH 6.0. T column was washed with the same buffer containing 4 M ur and proteins were eluted by a 2-liter linear gradient fr 300 mM to 1.5 M NaCl. The protein profile was determined the absorbance at 280 nm using an ISCO flowthrσugh monito Salt concentration was determined with a conductivity met (Radiometer Copenhagen) . The eluted fractions were assay as described above and fractions containing betaglycan we pooled and applied to a 25-ml wheat germ lectin colu (Pharmacia) equilibrated in 125 mM NaCl, 10 mM CHAPS, 1 glycerol, and 10 mM phosphate, pH 7.0. The column w washed with this buffer and bound protein was eluted in t same buffer containing 0.4 M N-acetyl-D-glucosamine.

The eluted sample was applied to a 1-ml TGF-βl affini column prepared from 1 mg porcine TGF-βl and Affigel (BioRad) according to the manufacturer's instructions. T column was washed with 125 mM NaCl, 10 mM CHAPS, 1 glycerol, and 10 mM phosphate, pH 7.0, followed by 500 NaCl, 10 mM CHAPS, 10% glycerol, and 10 mM phosphate, 5.0. Betaglycan was eluted with a 20-ml linear pH and sa gradient from the pH 5.0 buffer to a final buffer of 1 NaCl, 10 mM CHAPS, 10% glycerol and 10 mM phosphate, pH 2.0 Eluting fractions were collected into tubes containing 1/ volume 0.5 M phosphate, pH 7.0, and assayed for TGF binding activity as described below. For purification the core protein of betaglycan, wheat germ-purifi betaglycan was treated with heparitinase and chondroitinas ABC, as described below, before subsequent affini chromatography. Proteins were determined by the method of Bradford (1976) , by the BCA reagent method (Pierce) , or by amino acid analysis (Harvard Microchemistry Facility) , as appropriate. An estimate of the yield of betaglycan at each step was calculated based on the amount of TGF-β bound under standard assay conditions.

VISUALIZATION OF PURIFIED BETAGLYCAN. Betaglycan was visualized and purity assessed using ¹²⁵I-Bolton-Hunter reagent (Bolton and Hunter, 1973) . Bolton-Hunter reagent

(3 μCi/sample, DuPont/New England Nuclear) was dried under

N₂ and resuspended in 5 μl DMSO. The sample was added to the resuspended reagent and reactions were allowed to proceed for 16 hours at 4°C, then stopped by the addition of either electrophoresis sample buffer or Tris, pH 7.0, to a final concentration of 0.1 M. Samples were visualized by electrophoresis through 5-15% SDS-polyacrylamide gels and subsequent autoradiography of the fixed gels.

CHARACTERIZATION OF BETAGLYCAN. To identify the types of glycosaminoglycan chains present on betaglycan, affinity-labelled or Bolton-Hunter-labelled samples were incubated as previously described (Cheifetz et al., 1988b; Andres et al., 1989) with 2mIU of heparitinase/ml (ICN), 20 mU of chondroitinase ABC/ml (ICN) , or both enzymes for 16 hours at 37°C. Prior to enzyme addition, Bolton-Hunter labelled samples were centrifuged through 1 ml G-25 (Sigma) columns equilibrated in PBS/ 0.1% Triton X-100 (Sigma) to remove excess Bolton-Hunter reagent. Reactions were stopped by the addition of sample buffer before electrophoresis as described above. For treatment with endoglycosidase F (Boehringer Mannheim) , the radiolabelled band was excised from the fixed and dried gel and minced in Laemmli sample buffer and the enzyme (0.4 units/ml) was added as described previously (Cheifetz et al., 1988b). Samples were th electrophoresed again through 6% polyacrylamide gels and t bands visualized by autoradiography of the fixed and dri gels.

For liposome incorporation studies, purified betaglycan w affinity labelled with 150 pM ¹²⁵I-TGF-βl as describ above, concentrated through DEAE-Trisacryl, and eluted CHAPS containing buffer. Eluted samples were added to 0 mg of phosphatidyl choline (Sigma) dried in the bottom of glass test tube and l μCi ¹⁴C-dipalmitoyl phosphatid choline was added as a tracer. Samples were then dialyz against PBS overnight and liposomes separated centrifugation through a sucrose gradient as previous described (Andres et al., 1989). Core protein was similar treated, but the concentration step was omitted.

BETAGLYCAN MICROSEQUENCING. Betaglycan core prote destined for microsequencing was reduced a S-carboxyamidomethylated (Stone et al., 1989) by dissolvi it in 50 μl of 8 M urea, 0.4 M ammonium bicarbonat reducing it by addition of 5 μl of 45 mM dithiothreit followed by incubation at 50 °C for 15 in, and alkylati it by addition of 5 μl of 100 mM iodoacetamide followed incubation at room temperature for 15 min. This mixture w diluted 4-fold to a final buffer concentration of 2 M ure 0.1 M ammonium bicarbonate. Sequencing grade tryps (Boehringer Mannheim) was added to this solution to mainta an enzyme to substrate ratio of 25:1 (w/w) , and the mixtu was incubated at 37 °C for 16-20 h. The resulting pepti mixture was kept frozen at -20 °C until separation reverse phase HPLC. Tryptic betaglycan peptides were separated by chromatography on a Hewlett-Packard 1090 HPLC equipped with a 1040 diode array detector, using a Vydac 2.1 mm x 150 mm C18 column. The gradient employed was as previously described (Stone et al., 1989), with modifications. Briefly, a gradient of 5% B at 0 min, 33% B at 63 min, 60% B at 95 min and 80% B at 105 min was used where B was 0.055% trifluoroacetic acid in acetonitrile and the countergradient was 0.055% trifluoroacetic acid in water; the flow rate was 150 μl/min. Chromatographic data at 210 nm, 277 nm and 292 nm and UV spectra from 209-321 nm of each peak were obtained. While monitoring absorbance at 210nm, fractions were manually collected into 1.5-ml microcentrifuge tubes and immediately stored, without drying, at -20 °C in preparation for peptide sequence analysis.

Strategies for the selection of peptide fractions, and their subsequent automated Edman degradation, have been previously described (Lane et al., 1991). Samples were applied directly to a polybrene-precycled glass filter and placed in the reaction cartridge of an ABI Model 477A protein sequencer.

The reaction cartridge temperature was raised to 53 °C during coupling with a commensurate decrease in the three R2 delivery steps from 400 to 240 seconds. The resultant phenylthiohydantoin amino acid fractions were manually identified using an on-line ABI Model 120A HPLC.

BETAGLYCAN CDNA CLONING. For cDNA library construction, poly(A)⁺ RNA was isolated from Rat-1 cells using the Fast Track kit (Invitrogen) and used for the synthesis of random-primed cDNA (Gubler and Hoffman, 1983) . The double stranded cDNA, linked to EcoRI-NotI adapters, was ligated to EcoRI-digested lambda gtll arms (Huynh et al., 1985) a packaged in vitro using the Librarian XI kit (Invitrogen) order to create a bacteriophage library with a complexity 1.5xl0⁶ plaque forming units (pfu's)/mg cDNA.

For generation of betaglycan cDNA probes by PCR, ful degenerated primers were designed from the sequence of t CT-90 peptide (Fig. 8B) for PCR amplification against Rat-1 cDNA template (Saiki et al., 1988) . The sense prim (a 516-degeneracies 20-mer, designed from residues DQDLGFA) the antisense primer (a 1024-degeneracies 17-mer, design from residues YSNPDR) , as well as all other oligonucleotid were gel purified (Maxam and Gilbert, 1980) prior to us PCR was carried out in a volume of 50 ml using 10 Tris-Cl, pH 8.3, 50 mM KC1, 0.01% of gelatin, 1.5 MgCl₂, 0.2 of each dNTP, 1 mM of each primer (with tra amounts of end-labeled ³²P sense primer) and 5 mg/ml o cDNA. After 30 thermal cycles, each composed of 94 °C (6 sees) , 48 °C (60 sees) and 72 °C (60 sees) , the PC products were resolved in a denaturing polyacrylamide gel

Under these conditions only one PCR product with the correc predicted size (62 nt) was detected by autoradiography This product was purified from the gel, phosphorylated a its 5' ends, reannealed and subcloned at the Smal site o pGEM3Z (Promega) . The clones thus generated had 62bp-lon inserts which contained a unique sequence that encoded th residues AIQTCFLSPY of peptide CT-90. This unambiguou sequence was used in the design of the 29-mers jd4 and jd (sense and antisense orientations, respectively) .

For isolation of betaglycan cDNA clones, approximately 4.5x10^s unamplified clones from the Rat-1 cDNA library wer plated using E. coli strain Y1090 (30,000 pfu's/150 mm dish and lifted onto nylon membranes (Biotrans, ICN) in duplicates. The phage DNA was immobilized by UV-crosslinking (Stratalinker, Stratagene) and probed with ³²P end-labeled jd4 at 55 °C in 6x NET (900 mM NaCl, 90 mM Tris-Cl pH 8.0, 6 mM EDTA) , 0.1% SDS, 5x Denhart•s solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin) and 250 mg/ml of yeast RNA (Berent et al., 1985). Quick washes were done in 6x NET, 0.5% SDS at 37 °C, with a final wash (5-10 min in the same solution) at 55 °C. Six independent clones, lambda bg2 to lambda bg7, were detected in the primary screening, and were plaque-purified by two more rounds of screening.

Phage DNA prepared from these clones was digested with EcoRI; the released inserts were subcloned into the EcoRI sites of pGEM4Z and M13mpl9 for further analysis. The relative position of these plasmid subclone inserts and their 5' to 3 ^• orientation (with respect to betaglycan mRNA) were determined by PCR, primed with either the sense jd4 or the antisense jd3 primers and the SP6 promoter primer

(Promega) which hybridizes to sequences upstream of the multiple cloning site of pGEM4Z. PCR was done as described above, with the only difference that the 72 °C extension step was of 120 sees.

Only one combination of primers, either jd3/SP6 or jd4/SP6 produced one PCR product from each individual plasmid; from their sizes (determined in agarose gels) it was possible to deduce the orientation and the relative alignment of the six betaglycan cDNAs to each other. This alignment was confirmed upon sequencing. The lambda bg7 cDNA was completely sequenced using the chain termination method (Sanger et al., 1980) as available in the Sequenase kit (US Biochemicals) . Different templates were used for the sequencing: BamHI fragments subcloned in M13mpl8, th full-length insert subcloned at the EcoRI of the M13mpl9 and the nested deletions generated from the latter (Dale e al., 1985). Also, specific primers (Strauss et al., 1986 were used with the original mpl9 subclones. Betaglyca sequences were analyzed using the GCG sequence analysi software package (Devereux, et al., 1984) as available i the Columbia University Cancer Center Computer Facility.

CONSTRUCTION OF BETAGLYCAN EXPRESSION PLASMIDS. Expressio plasmids pCMV-bg (sense orientation) and pCMV-gb (antisens orientation) were constructed by subcloning the complet lambda bg7 cDNA insert into the EcoRI site of plasmid pCMV (Andersson et al., 1989). Orientation was confirmed b restriction analysis.

CELL TRANSFECTIONS. Monkey COS-1 cells (CRL1650) wer obtained from ATCC (American Type Culture Collection) . T cells (obtained from D. Livingston, Dana-Farber Cance Institute) are a CV-1P clonal line similar i characteristics to COS-l but having little or no endogenou betaglycan. Both cell lines were transfected with 2 μg/m of CsCl-purified plasmid diluted in Dulbecco's Modifie Eagles medium (DME) containing 10% NuSerum (Collaborativ Research) , 400 ug/ml of DEAE-dextran and 100 μM chloroquin as described by Seed and Aruffo (1987) . Twelve hours afte transfection, the cells were trypsinized and reseeded int 6-well or 12-well multicluster dishes for assay of TGF- binding 48-72 hours post transfection.

FBHE fetal bovine heart endothelial cells (CRL1395, ATCC) and L₆E₉ rat skeletal myoblasts (obtained from Nadal-Ginard, Harvard Medical School) seeded in 60-mm dishes were transfected using 10 μg of plasmid DNA and 30 μg of Lipofectin (GIBCO-BRL) according to manufacturer's instructions. The diluted DNA (in 750 μl of DME) was mixed with an equal volume of diluted Lipofectin and incubated for 15 min at room temperature prior to addition to the washed cell monolayer. After incubation at 37 °C for 3-5 h, the transfection mixture was replaced with 3 ml of the DMEM containing 10 % fetal bovine serum supplemented with 50 μg/ml endothelial cell mitogen (Bio edical Technologies Inc, Cambridge, MA) , for FBHE cells, or with 20 % fetal bovine serum, for L₆E_g cells, penicillin (100 units/ml), streptomycin (100 μg/ml each) and Fungizone (1:500 v:v, GIBCO) . The transfected cultures were processed as described above.

TGF-β BINDING AND AFFINITY LABELING ASSAYS. Monolayers were affinity-labeled with 100 pM ¹²⁵I-TGF-B1 as previously described (Massague, 1987) . To generate betaglycan core glycoprotein, cells were first incubated with heparitinase (0.5 mlU/ml) , chondroitinase ABC (50 mU/ml) , or a combination of the two enzymes for 3 hr at 37 °C (Cheifetz et al., 1988b). For competition studies, 50 pM ¹²⁵I-TGF-βl was used and unlabeled ligands were added at the start of the incubation. Affinity-labelled samples were visualized by electrophoresis through 5-8% polyacrylamide gels and subsequent autoradiography. To assay for the presence of soluble betaglycan, samples of media conditioned during days 2 and 3 post-transfeetion were applied to DEAE-trisacryl and affinity labelled with 150 pM ¹²⁵I-TGF-βl as previously described (Andres et al., 1989). Eluted samples were visualized as described above.

NORTHERN ANALYSIS. Pol (A)+ RNA from Rat-1, L6E9 and 3T3-L1 cell lines was separated in agarose gels containing formaldehyde (Rave et al., 1979) and transferred to Biotrans membranes by positive pressure blotting (Stratagene¹ Posiblot) . The complete 1BG7 cDNA insert was labelle with ³²P by random primed labelling (Amersham's Multiprim DNA labeling system) and used to probe the blotted membrane at 42 °C in 5x SSC (750 mM NaCl, 75 mM sodium citrate, p 7.0), 5x Denhart's solution, 50 mM sodium phosphate, 50 formamide, 0.1% SDS, and 100 mg/ml denatured sonicate salmon sperm DNA. Membranes were rapidly washed in 0.2x SS and 0.2% SDS at 37 °C followed by a final wash at 65 °C fo one hour in the same solution. The washed membranes wer then autoradiographed.

BINDING OF bFGF AND ITS ENZYMATICALLY GENERATED CORE PROTEI TO BETAGLYCAN. Betaglycan and its enzymatically generate core protein were purified from rat fetal tissue by combination of ion-exchange and TGF-βl affinit chromatography, electrophoresed through 6% polyacrylamid gels and electrotransferred to nitrocellulose. Afte transfer, the membranes were blocked with protein/detergent solution [3% bovine serum albumin, 0.1 Tween in phosphate-buffered saline] , probed with thi solution containing 0.6 nM bFGF (A gen) , labelled with ¹²⁵ using chloramine T, washed extensively with phosphate buffered saline, and visualized by autoradiography.

EFFECT OF bFGF ON THE MIGRATION OF BETAGLYCAN THROUG POLYACRYLAMIDE GELS. Fetal rat calvarial cells wer prepared as previously described (Noda, et al., 1987) an maintained in F12 medium with 10% FBS. Treatment with bFG was carried out in this medium. Cell surface TGF-β bindin proteins were detected by incubation of cells with 150 p ¹²⁵I-TGF-βl (R&D Systems) followed by cross-linking, a previously described (Cheifetz et al., 1987). Unlabelle TGF-βl was added to cells at the time of the addition o labelled TGF-βl. Labelled proteins were visualized by

electrophoresis on denaturing polyacrylamide gels an autoradiography.

LOSS OF HEPARAN SULFATE IN BETAGLYCAN FROM bFGF-TREATED CELLS. Osteoblasts treated with or without bFGF for 48 hours were affinity labelled with ¹²⁵I-TGF-βl and digested with heparitinase, chondroitinase ABC or with a mixture of the two enzymes. Triton X-100 extracts from affinity labelled cells were incubated with 2mIU of heparitinase/ l (ICN) , 20 lU of chondroitinase ABC/ml (ICN) , or both enzymes at 37°C, as previously described (Cheifetz, et al., 1988b) . Reactions were stopped by the addition of sample buffer before electrophoresis.

Examples

Example 1

Purification of betaglycan

Under the conditions employed for chromatography, betaglyca eluted from DEAE-trisacryl as a single diffuse peak a approximately 570 mM NaCl (Fig. 1), slightly later than th main peak of the retained protein. Most of the solubilize protein did not bind to DEAE at the pH and sal concentration used to load the column. Purification at thi step was approximately 100-fold. Application of elute betaglycan to wheat germ lectin-sepharose and subsequen elution with 0.4 M N-acetyl-D-glucosamine resulted in 50-fold purification at this step.

Purification of betaglycan to near homogeneity was obtaine by affinity chromatography of the wheat ger lectin-sepharose eluate in a TGF-βl-agarose column Non-specifically bound proteins were eluted by washing firs with phosphate-buffered saline, 10 m CHAPS, 10% glycerol, then with the same buffer containing 0.5 M NaCl at pH 5.0. Bound betaglycan was eluted with a linear gradient to 1 NaCl and pH 2.0 (Fig. 1). Some betaglycan could be elute by a pH 4.0 wash and material generated in this fashion wa used in subsequent competition studies, since the affinit of betaglycan for TGF-β was reduced by treatment with lowe pH. The final yield of bioactive betaglycan averaged 8%, based on the amount of TGF-β bound under standard assa conditions. The actual yield of purified protein may hav been somewhat higher, since betaglycan is known to los TGF-β binding activity after incubation below pH 4.0. Exa ple 2

Characterization of cell surface and tissue-derived betaglycan

Betaglycan affinity labelled by crosslinking to ¹²⁵I-TGF-B1 in solubilized rat embryo membranes had similar properties to betaglycan affinity labelling of Rat-1 cell monolayers (Fig. 2A) . Labelling with 50 pM ¹²⁵I-TGF-βl could be inhibited by the addition of competing native TGF-βl or TGF-β2 in the 0.2-5.0 nM range. Near-maximal competition was observed at approximately 5 nM TGF-βl or TGF-β2. TGF-B2 was somewhat more effective than TGF-βl as a competitor for betaglycan from Rat-1 cells but not from rat embryo membranes. Other labelled species were also observed in the rat embryo membrane samples (Fig. 2A) . These include a 70-kDa complex of bovine serum albumin present in the assays crosεlinked to ¹²⁵I-TGF-βl, and a 170-kDa labeled species which appeared to possess a lower affinity for TGF-β as compared to betaglycan since its labeling intensity was affected only by native TGF-β present at 5 nM or higher concentration.

Treatment of affinity-labeled extracts from Rat 1 cells with heparitinase, chondroitinase ABC, or a mixture of the two enzymes indicated that betaglycan in rat fibroblasts was a heparan sulfate/chondroitin sulfate proteoglycan with a 120-135 kDa core (Fig. 2B) . As shown in Figure 2B, the main product generated by the combined treatment of affinity-labeled rat embryo membrane extracts with heparitinase and chondroitinase had a similar migration through SDS-polyacrylamide gels as that from Rat 1 cells. Treatment of the rat embryo membrane material with each enzyme separately did not yield a lower molecular mass product suggesting that, like the molecule from Rat 1 cells betaglycan in solubilized membranes contained hepara sulfate and chondroitin sulfate GAG chains.

Example 3

Characterization of purified and affinity labelle betaglycan

Since betaglycan proved to be insensitive to traditiona protein staining methods, purity of eluted betaglycan wa assessed by labelling with ¹²⁵I-Bolton-Hunter reagent. Labelling using this procedure produced a diffuse ban similar to that observed by affinity labelling (Fig. 3B) . ¹²⁵I-Bolton-Hunter labelling tailed beyond the observe affinity labelling probably because, due to a pH-induce decrease in TGF-β affinity, betaglycan eluting at lower p could not be affinity-labelled as well as that eluting i the earlier fractions. Similar results were observed fo purification of the core protein, as shown in Figure 4.

Bolton-Hunter labelling was also used to compare th characteristics of the purified protein with those observe for the affinity-labelled molecule. Treatment of th ¹²⁵I-Bolton-Hunter labelled preparation with deglycosylatin enzymes confirmed the identification of this band as a single protein with the characteristics of betaglycan. Thus, treatment of the ¹²⁵I-Bolton-Hunter labelled purified protein with heparitinase, chondroitinase ABC, or a mixture of the two enzymes yielded a pattern of products very similar to that of betaglycan affinity-labelled in Rat-1 cells (compare Fig. 4A with Fig. 2B) . Furthermore, the 130-kDa betaglycan product obtained after removal of GAG chains was subsequently treated with endoglycosidase F. This treatment caused an increase in migration correspondin to loss of an apparent 10 kDa of N-linked carbohydrate (Fig 4B) , as had been previously reported for betaglyca affinity-labelled on cell surfaces (Cheifetz et al. , 1988b) . These results indicated that the purified betaglyca contained both heparan sulfate and chondroitin sulfate GA chains as well as N-linked glycans attached to a cor protein which migrated with an apparent molecular mass o 120 kDa.

Affinity-purified betaglycan had biochemical properties similar to the cell-surface molecule. Betaglycan eluted from the TGF-βl-agarose column by incubation with pH 4.0 buffer had an affinity for TGF-β comparable to that observed for betaglycan in Rat-l cells and solubilized rat embryo membrane preparations (Fig. 5) . The affinity of betaglycan eluted by the pH gradient for TGF-β was somewhat reduced, presumably due to partial denaturation resulting from elution at the lowered pH, but near maximal competition of ¹²⁵I-TGF-βl binding was still obtained with 10 nM TGF-βl or TGF-B2.

As has been shown previously (Andres et al., 1989), affinity-labelled betaglycan from cells is capable of incorporating into phosphatidylcholine liposomes. Similar results were obtained with purified betaglycan. Purified betaglycan was affinity labelled and mixed with phosphatidylcholine, and liposomes were formed by subsequent dialysis into non-detergent-containing buffer. The liposomes were separated from unincorporated material by centrifugation through a sucrose density gradient. As indicated in Figure 6, most of the betaglycan was found associated with liposomes at the top of the gradient, indicating that a membrane-anchoring region that was presen in betaglycan was retained during purification.

Example 4

Betaglycan cDNA Cloning

As a step towards molecularly cloning betaglycan, severa segments of internal amino acid sequence were obtained fro the purified protein. Since the intrinsic heterogeneity o betaglycan in its proteoglycan form (Cheifetz et al., 1988a; Segarini and Seyedin, 1988) made it a poor substrate fo microsequencing, we isolated betaglycan core glycoprotei devoid of GAG chains. This was accomplished by purifyin betaglycan from rat fetal tissue membranes through the DEA chromatography and wheat germ lectin chromatography steps described above. The fraction obtained after the second step was concentrated and digested with heparitinase and chondroitinase to remove GAG chains prior to chromatography over agarose-coupled TGF-βl (Andres et al., 1991). This step yielded a near-homogeneous preparation of the 120-kDa betaglycan core glycoprotein (Fig. 8A) that retained TGF-β binding activity and could be converted to a 110-kDa product by removal of N-linked carbohydrate with endoglycosidase F. Tryptic digestion of this betaglycan preparation and separation of the resulting fragments by reverse phase HPLC yielded 15 peptides that were suitable for N-terminal amino acid microsequencing. The sequences obtained from these peptides are presented in Fig. 9 (Seq. ID No. 1) .

Two pools of oligonucleotides were synthesized which included all possible sequences encoding the two ends of the betaglycan protein fragment sequence CT90 (Fig. 8B) (Seq. ID No. 2) . These oligonucleotide pools were designed in the sense and antisense orientations to serve as primers in a polymerase chain reaction (PCR) (Saiki et al., 1988). The template for this reaction was cDNA obtained by reverse transcription of Rat-1 fetal rat fibroblast poly(A)⁺ RNA. Rat-1 was the cell line of choice because it expresses betaglycan whose overall properties are very similar to those of betaglycan purified from fetal rat tissue (Andres et al., 1991). The resulting PCR product included a unique internal sequence which encoded CT90 (Fig. 8B; Seq. ID No. 2) . A ³²P-labeled synthetic oligonucleotide (jd4) corresponding to this sequence (Fig. 8B; Seq. ID No. 2) was used to screen 4.5 x 10⁵ recombinants from a random-primed Rat-1 fibroblast cDNA library cloned in lambda gtll. This screening yielded six positive clones whose inserts ranged in size from 1.0-3.9 kb. Sequencing of these cDNAs showed that they overlapped with each other (Fig. 8C) .

Sequencing of the largest cDNA insert, bg7, yielded a 3,931 bp sequence with an ATG codon starting at position 335 in a context favorable for translation initiation (Kozak, 1986) and followed by a 2,559 bp long open reading frame (Fig. 9; Seq. ID No. 1) . The inserts from two of the other positive cDNA clones were completely internal to bg7. The inserts from the other clones extended beyond bg7 into the 3 ' untranslated region which together with bg7 provided a 4.15 kb stretch of contiguous cDNA sequence (Fig. 8C) . The 3' untranslated region present in this sequence did not include a consensus polyadenylation signal and is, therefore, probably incomplete. The sequence of clone bg4 was completely divergent from that of the other clones in the region 5' to position 2174. This divergent region contained multiple stop codons and ended with a putative splice acceptor site. This region probably corresponds to an unspliced intron. Example 5

Betaglycan Protein Structure

The long open reading frame starting with nucleotide 335 in bg7 codes for a protein of 853 amino acids (Fig. 9; Seq. ID No. 1) . This predicted amino acid sequence has the features of a typical transmembrane protein starting with an amino terminal hydrophobic signal sequence followed, in order, by an extracellular region, a hydrophobic transmembrane region and a relatively short cytoplasmic region (Figs. 9 and 10; Seq. ID No. 1) . This orientation is inferred from the large reduction in the size of betaglycan caused by cell treatment with trypsin (Cheifetz et al., 1988b), and the presence of typical proteoglycan core protein features in the predicted extracellular domain (see below) .

According to established rules (von Heijne, 1988) , the putative signal sequence is tentatively assigned to include up to amino acid 24 (Fig. 9; Seq. ID No. 1). The betaglycan core protein devoid of a signal peptide has a calculated molecular mass of 91,643 daltons which is somewhat less than the value (110 kDa) estimated by SDS-PAGE of the purified (see Example 4 above) or affinity-labeled protein (Cheifetz et al., 1988b; Segarini and Seyedin, 1988). The heparan sulfate and chondroitin sulfate chains of proteoglycans are normally attached to the hydroxyl group of serine in the Ser-Gly sequence, particularly when this sequence is surrounded by acidic residues (Bourdon et al., 198" , There are six Ser-Gly sequences in the putative b&.~.aglycan extracellular domain. Sequences Ser⁵³⁵-Gly and Ser⁵⁴⁶-Gly are surrounded by acidic residues and are therefore the most likely sites for heparan sulfate or chondroitin sulfate attachment. The extracellular domain contains seven canonical sites for N-linked glycosylation of asparagin residues (Fig. 9; Seq ID No. 1) . Some of these sites ar probably glycosylated since betaglycan contain approximately 10 kDa of endoglycosidase-sensitive N-linke carbohydrate (Cheifetz et al., 1988b ; Andres et al., 1991). Most of these potential glycosylation and GAG attachmen sites are found in two clusters, one in the N-termina region and the other in the central region of the molecul (Fig. 10) .

The sixteen cysteines present in the extracellular domain of betaglycan are not arranged according to any known pattern. A proline-rich sequence (Pro-Ile-Pro-Pro-Pro-Pro) near the transmembrane region may act as a hinge at the base of the extracellular domain (Fig. 10) . Two potential cleavage sites are present near the transmembrane domain that if used would generate soluble forms of betaglycan such as those found in the media of various cell types (Andres et al., 1989) . One is the Lys⁷⁴⁵-Lys sequence that is cleaved by trypsin and might be a substrate for a cellular dibasic endoprotease (Barr, 1991) . The other is the Leu⁷⁵²-Ala-Val- Val sequence which is identical to a sequence in the membrane precursor for TGF-α (Derynck et al. , 1984; Lee et al. , 1985) that is cleaved by a highly regulated elastase-like activity that releases soluble TGF-α into the medium (Pandiella and Massague, 1991a; Pandiella and Massague, 1991b) .

The predicted cytoplasmic domain of betaglycan is only 43 amino acids long (Figs. 9 and 10; Seq. ID No. 1) . One distinctive feature of this domain is its high content (35%) of serines and threonines. Some of these residues might be sites for regulatory phosphorylations. In particular, threonine⁸¹⁷ in the juxtamembrane region o betaglycan is in a sequence that makes it a candidate for phosphorylation by protein kinas C (Pearson and Kemp, 1991) .

A search of the Genebank DNA databank (release 68.0) and th SwissProtein databank (release 17.0) indicated that the onl sequence with significant identity to betaglycan i endoglin. The amino acid sequences corresponding to th transmembrane and cytoplasmic regions of betaglycan an endoglin are strikingly similar with 63% identity and 92 similarity allowing for conservative substitutions (Fig. 11 Seq. ID. No. 3; Seq. ID No. 4). Endoglin, a major membran protein of human vascular endothelium, contains an RG sequence for potential recognition by cell adhesio receptors of the integrin family and is thought to play a important role in adhesion of endothelial cells to othe cells (Gougos and Letarte, 1990) . Rat betaglycan does no contain an RGD sequence and does not show similarity t endoglin in regions other than the transmembrane an cytoplasmic domain.

Example 6

Transmembrane and Soluble Betaglycans Are Encoded By Single cDNA

Confirmation that the bg7 cDNA encodes betaglycan wa obtained by expression of this cDNA in various mammalia cell lines. For these experiments, bg7 was subcloned in th sense (pCMV-bg) or anti-sense (pCMV-gb) orientation relative to the cyto egalovirus promoter present in th pCMV5 expression vector. The pCMV5 vector contains a vira SV40 origin of replication (Andersson et al., 1988) whic allows its amplification in monkey COS-1 cells. COS-1 cell transfected with pCMV-bg and subjected to TGF-βl bindin assays three days later showed a marked increase in specifi ¹²⁵I-TGF-B1 binding relative to controls transfected wit pCMV5 or pCMV-gb (see Table 1 below) .

Table 1.

¹²⁵I-TGF-βl BINDING TO CELL MONOLAYERS AND CONDITIONED MEDIA

Table 1 (continued)

Plasmids Transfected Cell Lines FBHE L6E9 cells media cells media ¹²⁵I-TGF-βl

ND

ND ND

- Cell l nes were transfected w t the ndicated plasmids. Two to three days post-transfection, cells were affinity-labelled with ¹²⁵I-TGF-βl and bound cpm extracted with Triton X-100. Data shown are corrected for non-specific binding measured in presence of a 100 fold excess of unlabeled TGF-βl except in b, where total cpm extracted into Triton X-100 are given. Conditioned media were assayed for presence of soluble TGF-β-binding proteoglycans and the cpm listed represent the material produced by the cell monolayers which were used for the affinity-labelling assay. ND: Not determined. In order to detect individual cell surface TGF-β receptors, COS-1 cell transfectants were crosslinked to cell-bound ¹²⁵I-TGF-B1 by addition of disuccinimidyl suberate. Analysis of extracts from these cells by SDS-PAGE revealed increased labelling of a broad 300-kDa component in cells transfected with pCMV-bg (Fig. 12A) . This component was not detectable in similar autoradiographic exposures of COS-1 cell transfected with pCMV5 or pCMV-gb (Fig. 12A) but comigrated with the endogenous betaglycan present in these cells as detected by prolonged exposure of the gels. The 300-kDa component was sensitive to heparitinase and chondroitinase ABC, and could be converted to a heterogeneous core product of 120-140 kDa by co-incubation with both enzymes (Fig. 12A) . The core product generated by treatment of cell monolayers with these enzymes could still bind ¹²⁵I-TGF-βl (Fig. 12A) as has been demonstrated for endogenous betaglycan (Cheifetz et al., 1988b; Segarini and Seyedin, 1988; Andres et al., 1991). Moreover, ¹²⁵I-TGF-βl binding to this proteoglycan was competed by unlabelled TGF-βl and H2 (Fig. 12B) . All these properties are typical of betaglycan from various cell lines and tissue sources (Cheifetz et al., 1987; Cheifetz et al., 1988b; Segarini and Seyedin, 1988; Cheifetz and Massague, 1989; Andres et al., 1991) . We concluded that bg7 encodes betaglycan.

pCMV-bg but not pCMV-gb also directed the synthesis of betaglycan when expressed in FBHE fetal bovine heart endothelial cells (Fig. 12C) , Tl monkey kidney cells and L₆E₉ rat skeletal yoblasts. These three cells lines do not express endogenous betaglycan although they express TGF-β receptors I and II, as detected by affinity labelling (Massague et al 1986; Cheifetz et al., 1990; Fig. 12C) . A substantial portion of the betaglycan core protei overexpressed in FBHE cells reached the cell surface as cor protein lacking GAG chains but capable of binding TGF-β (Fig. 12C) .

Various cell lines that express membrane-bound betaglyca also release soluble betaglycan into the medium (Andres e al., 1989). To determine if the soluble form of betaglyca is encoded by the same cDNA as the membrane-bound form, th media from cells transfected with various plasmids wer collected and fractionated over DEAE-trisacryl in order t obtain a proteoglycan-rich fraction. The ¹²⁵I-TGF-βl binding activity was much higher in the soluble proteoglyca fraction from cells transfected with pCMV-bg than from cells transfected with control plasmids (see Table 1) .

Crosslinking experiments confirmed the presence of a high level of soluble betaglycan in the media from pCMV-bg transfected cells compared to the corresponding pCMV-gb transfectants (Fig. 13) . Thus, a single cDNA can direct the synthesis of membrane-bound and soluble betaglycan forms.

Example 7

Northern Analysis of Betaglycan mRNA

The bg7 cDNA was used as a probe to determine the size and distribution of betaglycan mRNA in various cell lines. Northern assays of poly(A)⁺ RNA from Rat-1 fibroblasts and mouse 3T3-L1 fibroblasts indicated the presence of a single hybridizing species of approximately 6 kb (Fig. 14) . No signal was detected in L₆E₉ rat skeletal myoblasts, a cell line that does not express detectable betaglycan protein (Massague et al. , 1986) . Example 8

Binding of bFGF to betaglycan

Purified betaglycan was electrophoresed on a denaturin polyacrylamide gel, electrotransferred to a nitrocellulos sheet and probed with ¹²⁵I-bFGF. Intact betaglycan boun ¹²⁵I-bFGF. Predigestion with heparitinase an chondroitinase to generate GAG-free core protein eliminate the ¹²⁵I-bFGF binding activity (see Fig. 15) . Thi treatment did not affect the TGF-β binding activity o betaglycan. Loss of ¹²⁵I-bFGF binding activity was als observed after digestion of betaglycan with heparitinas alone.

Example 9

Regulation of betaglycan by TGF-β or bFGF

Calvaria osteoblasts treated with InM TGF-β for up to 48 hours showed no change in the structure of betaglycan usin ^{1 5}I-TGF-βl as an affinity labelling probe, and showed a slight decrease in betaglycan labelling that was probably due to bound TGF-β carried over from pre-incubation. However, a clear shift in mobility was observed i osteoblasts treated with bFGF (see Fig. 16) . Betaglycan from control osteoblasts migrated on denaturing gels as a broad labelled species of over 300 kDa (see Fig. 16) , as is characteristic of betaglycan from other cell types (Cheifetz et al., 1987; Segarini and Seyedin, 1988; Cheifetz et al., 1988b; Cheifetz and Massague, 1989) . Treatment of osteoblasts with bFGF for 48 hours increased the mobility of betaglycan, which appeared as a species of about 250 kDa (see Fig. 16) . This change did not decrease the TGF-β binding activity of betaglycan. No change was observed i the mobility of TGF-β receptors I and II (see Fig. 16) , two membrane proteins involved in TGF-β signal transduction. The effect of bFGF on betaglycan mobility was time-dependent (^tι/₂ ^{= 4} k°^urs) ^and concentration-dependent (ED₅₀ - 0.1 nM bFGF) . Similar, but less dramatic, effects of bFGF on betaglycan were observed in Fl (fibroblast-enriched) primary cultures of rat calvaria and in the MC 3T3E1 mouse calvaria osteoblast cell line, but not in the ROS 17/2.8 rat osteoma cell line, MVlLu mink lung epithelial cells or Rat-1 embryo fibroblasts.

Example 10

Nature of the modification of betaglycan induced by bFGF

Affinity labelled-betaglycan from bFGF-treated or untreated cultures was digested with heparitinase or chondroitinase ABC. Digestion of betaglycan with a combination of both enzymes generated a set of 120-130 kDa core proteins that was not affected by cell treatment with bFGF (see Fig. 17) . Digestion with heparitinase alone also showed no change due to bFGF (see Fig. 17) . In addition, it showed that rat calvaria osteoblasts contained a mixed betaglycan population, with forms that resisted complete removal of GAGs by heparitinase and thus contained chondroitin sulfate (BG-C forms in Fig. 17) , and forms that yielded free core protein and thus contained exclusively heparitinase- sensitive GAG chains.

Digestion of betaglycan with chondroitinase generated a clearly different set of patterns from control and bFGF- treated cells. Chondroitinase converted betaglycan from control cells into two products, one of over 200 kDa and thus with a high heparan sulfate content (BG-HH in Fig. 17) and one of approximately 140 kDa and thus with a very l heparan sulfate content (BG-LH in Fig. 17) . Osteoblast treated with bFGF presented only this form of betaglycan No change was seen in the level or the electrophoreti mobility of the major cell-associated proteoglycans a determined by metabolic labelling of rat calvari osteoblasts with ³⁵S-sulfate after bFGF treatment suggesting that bFGF did not affect cell surface proteins i general. It remains to be determined if bFGF acted b altering GAG synthesis or expression of betaglycan cor proteins that are poor acceptors of heparan sulfate chains

Discussion

Purification and Characterization of Betaglycan Protein

The examples presented here describe the purification of the TGF-β binding proteoglycan betaglycan from fetal rat tissue. Purified betaglycan has the same properties as those described for betaglycan identified on the surface of cells and in solubilized membranes. The purified molecule retains a high affinity for TGF-β and the nature of its carbohydrate chains is similar to that of betaglycan found in embryonic rat fibroblasts and in various other cell lines (Cheifetz et al., 1988b; Segarini and Seyedin, 1988; Andres et al., 1989) . It also maintains the ability to incorporate into liposomes, indicating that this property is intrinsic to betaglycan rather than due to interaction with an accessory protein. Although betaglycan is highly sensitive to proteolysis (Cheifetz et al. , 1988b) , the yield of purified betaglycan and the properties of its core protein indicate that extensive proteolysis did not occur during purification using this protocol.

The purified molecule therefore possessed the characteristics of native membrane-bound betaglycan . It is not clear why the core protein of betaglycan affinity labelled on the cell surface migrates on electrophoresis gels as a two or more bands (Figure 2; Cheifetz et al. , 1988b; Cheifetz and Massague, 1989; Andres et al., 1989) whereas the purified betaglycan core migrates as a single band. The various forms observed by affinity labeling of cell surface betaglycan may represent different conformations of the cross-linked betaglycan-TGF-β complex, partial loss of one TGF-β monomer after reduction preceding electrophoresis, different conformational changes induced by the presence of other proteins in the membrane, or possibl alternatively forms of betaglycan some of which are los during purification. Some of these potential variables migh also determine why TGF-B2 was somewhat more effective tha TGF-βl as a competitor for betaglycan from intact Rat- cells and affinity-purified betaglycan, but not betaglyca from rat embryo membranes. Varying differences in affinit of betaglycan for distinct TGF-β isoforms have also bee observed between different cell types (Cheifetz and Massague, 1989; Segarini et al., 1987).

It has been suggested that at least one other proteoglycan, the secreted protein decorin, may also bind TGF-β (Yamaguchi et al., 1990). Since decorin is a major secretory proteoglycan that is deposited into extracellular matrices, it might be expected to be present as a contaminant in crude membrane preparations like those used for the purification of betaglycan. Even though betaglycan is a minor component of the cell membranes (estimated average 10,000 copies/cell) , we have found no evidence for the presence of decorin or any proteoglycan other than betaglycan in our affinity-purified fractions. Purified betaglycan does not resemble other proteoglycans whose purification has been reported (Yamaguchi et al., 1990; Sant et al., 1985; Fisher et al., 1989; Schmidt and Buddecke, 1989; Ecarot-Charrier and Broekhuyse, H. , 1987; DeBoeck, et al., 1987; Heremans et al., 1988; David et al., 1990; Jalkanen et al., 1988; Saunders et al., 1989) and the N-terminal amino acid sequence of multiple tryptic fragments obtained frcn its core protein does not match any previously described protein sequence. Availability of the purified protein retaining a high affinity for TGF-β and the hydrophobic characteristics of the cell-surface molecule will facilitate investigation of its role in TGF-β function and its involvement i interactions with other growth factors.

Betaglycan Structure

The predicted betaglycan core protein is synthesized as a 853 amino acid transmembrane protein with a conventional signal sequence at the N-terminus and a single transmembrane domain near the C-terminus leaving a relatively large extracellular domain and a short (43 amino acid) cytoplasmic tail. This overall domain structure is similar to those of other membrane proteoglycan core proteins including syndecan (Saunders et al. , 1989) , the CD44 lymphocyte homing receptor (Stamenkovic et al., 1989; Goldstein et al., 1989), thrombomodulin (Dittman et al., 1988) and a heparan sulfate proteoglycan of human lung fibroblasts (Marynen et al., 1989) . However, the amino acid sequence of betaglycan is unrelated to the sequences of these other membrane proteoglycans.

Betaglycan is found in animal tissues and cultured cells in a form containing both chondroitin sulfate and heparan sulfate GAGs (Cheifetz et al., 1988b; 1989; Segarini and

Seyedin, 1988; Andres et al 1991) . Chondroitin sulfate chains, and probably heparan sulfate chains, are attached to the free hydroxyl group of Ser-Gly sequences in the proximity of acidic residues (Bourdon et al., 1987). There are two such sequences in the betaglycan core, both of them clustered with several potential N-linked glycosylation sites and two other Ser-Gly sequences in the middle of the protein. Two additional ser-gly sequences without neighboring acidic residues are located in the N-terminal region. An unexpected finding of the present studies is the hig degree of similarity between the transmembrane an cytoplasmic regions of betaglycan and endoglin. Endoglin i a membrane protein of endothelial cells that exists as disulfide-linked di er of two identical 95 kd subunits whos extracellular domain contains an RGD sequence (Gougos an Letarte, 1990) . Since the RGD sequence is a site fo recognition by cell adhesion receptors of the integri family (Ruoslahti and Pierschbacher, 1987) , it is possibl that endoglin may be involved in cell-cell recognitio (Gougos and Letarte, 1990) . The sequences of th extracellular regions of betaglycan and endoglin are totall unrelated and betaglycan contains no RGD sequence, whic suggests that their similar transmembrane and cytoplasmi domains might have a common, as yet unknown importan function.

An unusual feature of the cytoplasmic tail is its high (35%) serine and threonine content. Threonine⁸¹⁷ in particular i surrounded by a sequence that makes it a probable substrat for protein kinase C (Pearson and Kemp, 1991) , and it location in the juxtamembrane region of betaglycan i analogous to the location of Thr⁶⁵⁴ in the epidermal growt factor receptor, a known site for regulation of thi receptor by protein kinase C (Hunter et al., 1984). It is not yet known if betaglycan is regulated by protein kinase C activators.

The results of betaglycan transfection experiments indicate that the polymorphism of betaglycan is not necessarily due to the existence of multiple betaglycan genes or multiple transcripts. The present results show that a single cDNA can direct the synthesis of a membrane-bound form of betaglycan whose core glycoprotein appears electrophoretically heterogeneous, a form of betaglycan tha is released into the medium, and a membrane-bound form o betaglycan devoid of GAG chains. Released forms have als been described for the membrane proteoglycan syndecan (Jalkanen et al., 1987; Weitzhandler et al., 1988).

The soluble form of betaglycan is probably generated, at least in part, by cleavage of the membrane-bound form. A potential cleavage site that would release an almost complete betaglycan ectodomain into the medium is a Lys-Lys sequence near the transmembrane region. Another is the sequence Leu-Ala-Val-Val which is identical to the sequence in the membrane precursor for TGF-α that is cleaved to generate soluble TGF-α (Derynck et al. , 1984; Lee et al., 1985) . Cleavage of this sequence in proTGF-α occurs at the cell surface through a process that is rapidly activated by phorbol esters, calcium ionophores, platelet-derived growth factor and other serum factors (Pandiella and Massague, 1991a; 1991b) and could also act on other membrane proteins such as betaglycan.

Implications For Function

What role betaglycan plays as a mediator of cellular interaction with TGF-β is unknown at the moment, but at least three functions can be suggested based on the structural properties of betaglycan and its high affinity for TGF-β.

First, betaglycan could be involved in capturing and retaining TGF-β from the pericellular environment for presentation to the signaling receptors. After its synthesis and secretion, TGF-β goes through an unusually complex set of events before it appears in the active form (Pircher et al., 1986; Lyons et al., 1988; Miyazono an Heldin, 1989; Sato and Rifkin, 1989; Kanzaki et al., 1990 Dennis and Rifkin, 1991) , and the active form can be ver rapidly cleared if it remains free (Coffey et al., 1987) In order for betaglycan to effectively capture activate TGF-β from the pericellular environment for presentation t the signaling receptors, betaglycan should have lowe affinity for TGF-β than the signaling receptors and be mor abundant than those receptors. These two criteria seem t be fulfilled. Thus, although the affinity of betaglycan fo TGF-β is high (K_D = 3-30 x 10^"11 M) , it is somewhat lowe than that of the putative TGF-β signaling receptors I and I (K_D 5-30 x 10^"12 M) (Massague et al. , 1990). In many cel types, betaglycan is more abundant (1-10 x 10 molecules/cell) than receptors I and II (1-50 x 10 molecules/cell) .

Also related to this potential role, the heparan sulfat chains of betaglycan might act as binding sites fo presentation of FGF to its receptors, as shown by th ability of basic FGF to bind to the heparan sulfate chain of purified betaglycan. Binding of FGF to heparan sulfat appears to be indispensable for its binding to the hig affinity FGF signaling receptors (Yayon et al., 1991) an for biological responses to FGF (Rapraeger et al., 1991) Betaglycan might be one of the proteoglycans that contribut to this function in the cell, thus serving simultaneously a a component of the TGF-β and FGF receptors systems. The GA chains of betaglycan are dispensable for TGF-β binding o cell surface exposure of betaglycan allowing for independen regulation of the TGF-β and FGF binding functions (Cheifet and Massague, 1989; Andres et al., 1991). The level an composition of the GAG chains in membrane proteoglycans ca be regulated by factors in the pericellular environment For example, tissue location or cell treatment with TGF- affects the GAG chain composition of syndecan (Sanderson an Bernfield, 1988; Rasmussen and Rapraeger, 1988), an treatment with bFGF markedly decreases the proportion o heparan sulfate GAG attached to betaglycan in osteoblasts.

A second potential role of betaglycan which is in contras with the first one is that betaglycan could act to restrai TGF-β action by depleting this factor from the medium. This interesting function has been proposed for decorin, a small proteoglycan that is abundant in extracellular matrices and can bind TGF-β albeit with lower affinity and binding capacity than betaglycan (Yamaguchi et al., 1990). Overexpression of betaglycan did not appear to alter the level of TGF-βl bound to receptors I and II in FBHE and L₆E₉ cells. However, the use of a single concentration of ¹²⁵I-TGF-βl in our present binding assays may not be adequate to detect whatever positive or negative effect the overexpression of betaglycan might have on TGF-β binding to the other receptors. Stable cell clones overexpressing betaglycan will be generated to address this question.

Although the two latter roles assume that betaglycan lacks the ability to signal TGF-β responses, a role of betaglycan in signaling cannot be discarded as yet. The arguments that can be mounted against such a role are based on the apparent absence of betaglycan in some myoblast, hematopoietic and endothelial cell lines which respond to TGF-β (Massague et al. , 1986; Ohta et al., 1987; Segarini et al., 1989; Cheifetz et al. , 1990) and the lack of known structural features in the short cytoplasmic tail of betaglycan that could suggest a signaling function. However, betaglycan could be involved in signaling by associating with other components of the receptor system as occurs with certain cytokine binding proteins that have very short cytoplasmi tails (Hatakeyama et al., 1989; Taga et al., 1989), and th involvement of betaglycan in signaling could vary dependin on the cellular context such as appears to occur with th p75 protein of the NGF receptor system (Hempstead et al. 1991; Cordon-Cardo et al., 1991).

The observed release of betaglycan ectodomain from the cel surface could dramatically alter any of the function described above. Release would allow betaglycan t accumulate in the extracellular matrix where it could act a a general reservoir of TGF-β for surrounding or migratin cells, or as a soluble inhibitor of TGF-β. The possibilit that the proteolytic release of betaglycan might b regulated by certain physiological and pharmacologica agents, as in the case of the membrane TGF-α precurso (Pandiella and Massague 1991a; 1991b) , is of interest.

It is likely that betaglycan is involved in at least one an possibly several of the functions mentioned above, question that can be clarified through the study of th betaglycan protein and the manipulation of its gene a facilitated by the availability of a betaglycan cDNA clone.

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Claims

What is claimed is:

1. A purified betaglycan protein.

The purified betaglycan protein of claim 1, wherei the protein is a mammalian protein.

3. A mammalian protein of claim 2, wherein th mammalian protein is a murine protein.

4. A mammalian protein of claim 2, wherein th mammalian protein is a human protein.

The purified betaglycan protein of claim 1, capabl of binding a molecule of the transforming growt factor TGF-β and a molecule of basic fibroblas growth factor.

6. The purified betaglycan protein of claim 1, capabl of binding a molecule of the transforming growt factor TGF-β.

7. The purified betaglycan protein of claim 1, capabl of binding a molecule of basic fibroblast growt factor.

A nucleic acid molecule encoding the protein o claim 1.

The nucleic acid molecule of claim 8, wherein th nucleic acid molecule is a DNA molecule.

10. A DNA molecule of claim 9, wherein the DNA molecul is a cDNA molecule.

11. A purified, soluble betaglycan protein.

12. The soluble betaglycan protein of claim 11, capable of binding a molecule of the transforming growth factor TGF-β and a molecule of basic fibroblast growth factor.

13. The soluble betaglycan protein of claim 11, capable of binding a molecule of the transforming growth factor TGF-β.

14. The soluble betaglycan protein of claim 11, capable of binding a molecule of basic fibroblast growth factor.

15. A nucleic acid molecule encoding the soluble betaglycan protein of claim 11.

16. The nucleic acid molecule of claim 15, wherein the nucleic acid molecule is a DNA molecule.

17. The DNA molecule of claim 16, wherein the DNA molecule is a cDNA molecule.

18. The soluble betaglycan protein of claim 11 labelled with a detectable marker.

19. The soluble betaglycan protein of claim 18, wherein the detectable marker is a radioactive isotope, enzyme or dye.

20. The soluble betaglycan protein of claim 11 free of glycosaminoglycan chains.

21. The glycosaminoglycan-free soluble protein of clai 20 capable of binding a molecule of th transforming growth factor TGF-β.

22. The glycosaminoglycan-free soluble protein of clai 20 labelled with a detectable marker.

23. The glycosaminoglycan-free soluble protein of clai 22, wherein the detectable marker is a radioactiv isotope, enzyme or dye.

24. A protein complex comprising the soluble betaglyca protein of claim 11, wherein the soluble protein i bound to a molecule of the transforming growt factor TGF-β and a molecule of basic fibroblas growth factor.

25. A protein complex comprising the soluble betaglycan protein of claim 11, wherein the soluble protein is bound to a molecule of the transforming growth factor TGF-β.

26. A protein complex comprising the soluble betaglycan protein of claim 11, wherein the soluble protein is bound to a molecule of basic fibroblast growth factor.

27. A protein complex comprising the glycosaminoglycan- free soluble protein of claim 20, wherein the glycosaminoglycan-free protein is bound to a molecule of the transforming growth factor TGF-β.

28. A pharmaceutical composition which comprises the soluble betaglycan protein of claim 11 and a pharmaceutically acceptable carrier.

29. A pharmaceutical composition which comprises the glycosaminoglycan-free protein of claim 20 and a pharmaceutically acceptable carrier.

30. A pharmaceutical composition comprising the protein complex of claim 24 and a pharmaceutically acceptable carrier.

31. A pharmaceutical composition comprising the protein complex of claim 25 and a pharmaceutically acceptable carrier.

32. A pharmaceutical composition comprising the protein complex of claim 26 and a pharmaceutically acceptable carrier.

33. A pharmaceutical composition comprising the protein complex of claim 27 and a pharmaceutically acceptable carrier.

34. A pharmaceutical composition comprising the nucleic acid molecule of claim 8 and a pharmaceutically acceptable carrier.

35. A pharmaceutical composition comprising the nucleic acid molecule of claim 15 and a pharmaceutically acceptable carrier.

36. A monoclonal antibody specifically reactive with the purified betaglycan protein of claim 1.

37. A monoclonal antibody of claim 36, labelled with detectable marker.

38. A monoclonal antibody of claim 37, wherein th detectable marker is a radioactive isotope, enzym or dye.

39. A hybridoma cell line producing the monoclona antibody of claim 36.

40. A monoclonal antibody specifically reactive wit the soluble betaglycan protein of claim 11.

41. A monoclonal antibody of claim 40, labelled with detectable marker.

42. A monoclonal antibody of claim 41, wherein th detectable marker is a radioactive isotope, enzym or dye.

43. A hybridoma cell line producing the monoclona antibody of claim 40.

44. A pharmaceutical composition comprising th monoclonal antibody of claim 36 and pharmaceutically acceptable carrier.

45. A pharmaceutical composition comprising th monoclonal antibody of claim 40 and pharmaceutically acceptable carrier.

46. A gene transfer vector which comprises the nuclei acid molecule of claim 8.

47. A gene transfer vector of claim 46 which comprises a plasmid.

48. A gene transfer vector of claim 46 which comprises a virus.

49. A host vector system for the production of the betaglycan protein, which comprises the gene transfer vector of claim 46 in a suitable host.

50. The host vector system of claim 49, wherein the suitable host is a eukaryotic cell.

51. The host vector system of claim 50, wherein the eukaryotic cell is a yeast.

52. The host vector system of claim 50, wherein the eukaryotic cell is a mammalian cell.

53. A gene transfer vector which comprises the nucleic acid molecule of claim 15.

54. A gene transfer vector of claim 53 which comprises a plasmid.

55. A gene transfer vector of claim 53 which comprises a virus.

56. A host vector system for the production of the soluble betaglycan protein, which comprises the gene transfer vector of claim 53 in a suitable host. -81-

57. A host vector system of claim 56 suitable for t production of the glycosaminoglycan-free solub betaglycan protein, wherein the suitable host ce is defective in glycosaminoglycan synthesis.

58. The host vector system of claim 56, wherein t suitable host is a eukaryotic cell.

59. The host vector system of claim 58, wherein t eukaryotic cell is a yeast.

60. The host vector system of claim 58, wherein t eukaryotic cell is a mammalian cell.

61. A method of producing a purified betaglycan prote which comprises growing the host vector system claim 49 under conditions permitting production betaglycan protein and recovering the betaglyc protein so produced.

62. The protein produced by the method of claim 61,

63. A method of producing a soluble betaglycan prote which comprises growing the host vector system claim 56 under conditions permitting production the soluble betaglycan protein and recovering t soluble betaglycan protein so produced.

64. The protein produced by the method of claim 63.

65. A method of producing a glycosaminoglycan-fr soluble betaglycan protein which comprises growi the host vector system of claim 57 under conditio permitting production of the glycosaminoglycan-fr soluble betaglycan protein and recovering th glycosaminoglycan-free soluble betaglycan protei so produced.

66. The protein produced by the method of claim 65.

67. A method of purifying the protein of claim 1 comprising: (i)solubilizing cell and tissue membranes containing the betaglycan protein;

(ii)passing a sample of said solubilized ebrane through an ion-exchange chromatography column; (iii)passing the resulting eluate fractions containing the betaglycan through a lectin chromatography column;

(iv)passing the resulting eluate fractions containing the betaglycan through a column containing immobilized TGF-β and recovering the betaglycan from the resulting eluate.

68. The protein produced by the method of claim 67,

69. A method of purifying the protein of claim 20 which comprises contacting a sample of the purified, soluble betaglycan protein with an amount of an enzyme composition effective to cleave the glycosaminoglycan chains from the core of the soluble betaglycan protein.

70. A method of claim 69, wherein the pharmaceutical composition comprises heparitinase and chondroitinase.

71. A method of claim 69, wherein the enzyme composition comprises heparitinase.

72. A method of claim 69, wherein the enzy composition comprises chondroitinase.

73. The protein produced by the method of claim 71.

74. A method of determining the ratio of active TGF to the total amount of TGF-β in the body of animal which comprises:

(i) isolating a suitable sample from the body of t animal;

(ii)contacting the sample with an amount of t pharmaceutical composition of claim 28 or 2 effective to bind all the active TGF-β in th sample, under conditions suitable to the formatio of complexes between soluble betaglycan protein an active TGF-β;

(iii)determining the amount of complex formed; an (iv)determining the total amount of TGF-β in th sample and comparing the total amount of TGF-β i the sample to the amount of complex formed in th sample.

75. A method of claim 74, wherein the suitable sampl is blood.

76. A method of claim 74, wherein the suitable sampl is body tissue.

77. A method of claim 74, wherein the animal is a rat

78. A method of claim 74, wherein the animal is human.

79. A method of increasing the concentration of fre TGF-β in the body of an animal which comprise administering to the animal an amount of the pharmaceutical composition of claim 28, 29, 30, 31 or 33 effective to increase the concentration of free TGF-β in the body of the animal.

80. A method of claim 79, wherein the animal is a rat.

81. A method of claim 79, wherein the animal is a human.

82. A method of decreasing the concentration of free TGF-β in the body of an animal which comprises administering to the animal an amount of the pharmaceutical composition of claim 28 or 29 effective to decrease the concentration of free TGF-β in the body of the animal.

83. A method of claim 82, wherein the animal is a rat.

84. A method of claim 82, wherein the animal is a human.

85. A method of imaging TGF-β in the body of an animal which comprises:

(i)administering to the animal an amount of the pharmaceutical composition of claim 28 or 29 effective to bind TGF-β throughout the body of the animal, under conditions permitting the formation of complexes between TGF-β and betaglycan; (ii)administering to the animal an effective imaging amount of the pharmaceutical composition of claim 44 or 45 under conditions suitable for the formation of complexes between the monoclona antibody and the betaglycan protein, (iii) clearing any uncomplexed imaging agent fro the body of the animal; and (iv) imaging any monoclonal antibody-betaglyca protein complexes found in the body of the animal.

86. A method of claim 85, wherein the animal is human.

87. A method of increasing the concentration of fre fibroblast growth factor in the body of an anima which comprises administering to the animal a amount of the pharmaceutical composition of clai 28, 30 or 32 effective to increase the concentration of free fibroblast growth factor i the body of the animal.

88. A method of claim 87, wherein the animal is a rat.

89. A method of claim 87, wherein the animal is a human.