Domain structure of the basement membrane heparan sulfate proteoglycan

We have used proteolytic digestions and immunological reactivity to map regional domains of the 400-kilodalton (kDa) core protein of the heparan sulfate containing basement membrane proteoglycan from the Englebreth-Holm-Swarm tumor. Digestion with V8 protease caused the rapid release of numerous large peptides ranging in size from 80 to 200 kDa and a 44-kDa peptide. The 44-kDa peptide (P44) was stable to further digestion, but the larger peptides were eventually degraded to a 46-kDa peptide (P46). Both the P44 and P46 fragments migrate slower in the presence of a reducing agent, indicating intrachain disulfide bonding, and do not have heparan sulfate side chains. Antisera to the P46 fragment, however, did not react with P44 fragment, and the amino acid compositions of P46 and P44 fragments were different. This suggests that these two fragments were unrelated. Trypsin digestion of the proteoglycan immediately released a 200-kDa peptide (P200) that also lacked heparan sulfate side chains. Digestion of the P200 fragment with V8 protease produced the P44 and P46 fragments in the same temporal sequence seen with V8 protease digestion of the proteoglycan. Antisera to the P200 fragment reacted strongly with the P44 and P46 fragments. These results show that the P44 and P46 domains are contained within the P200 domain. The rapid release of the P44 domain indicates that it is located at one end of the core protein. The large size of these proteolytic fragments suggests the core protein contains considerable conformational structure, and the absence of heparan sulfate on the P200 domain indicates that the side chains are asymmetrically located on the core.     

Comparisons of antibody reactivity and enzyme sensitivity between small proteoglycans from bovine tendon, bone, and cartilage

Preparations of small proteoglycans from bovine tendon, bone, and cartilage have been compared for sensitivity to various enzymes and reactivity with different polyclonal antibodies. Chondroitinase ABC digestion of all proteoglycans generated a core protein preparation that migrated similarly in sodium dodecyl sulfate-polyacrylamide electrophoresis as a doublet band with Mr approximately equal to 45,000. The small proteoglycans of cartilage were divided into two populations based upon electrophoretic migration of the intact molecules (Rosenberg, L. C., Choi, H. U., Tank, L-H., Johnson, T. L., Pal, S., Webber, C., Reiner, A., and Poole, A. R. (1985) J. Biol. Chem. 260, 6304-6313). The core preparations of tendon, bone, and the faster-migrating (PG II) proteoglycans of cartilage all interacted in Western blot/enzyme-linked immunosorbent assay analysis with polyclonal antibody raised against either the tendon or bone proteoglycans. The slower-migrating (PG I) proteoglycans of cartilage did not react with these antibodies. Digestion of the tendon small proteoglycan with Staphylococcus aureus V8 protease released glycosaminoglycan chains from the molecule and generated a 40-kDa protein fragment that was resistant to further rapid degradation by the enzyme. This large digestion fragment was also prominent following V8 protease digestion of the faster-migrating (PG II) population of small cartilage proteoglycans, but not the small proteoglycan of bone. The N-terminal amino acid sequence of the tendon (PG II) proteoglycan was determined. These observations provide additional evidence for heterogeneity among the chemically similar small proteoglycans from different tissues.     

Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon.

The small dermatan sulphate proteoglycan of bovine tendon demonstrated a unique ability to inhibit fibrillogenesis of both type I and type II collagen from bovine tendon and cartilage respectively in an assay performed in vitro. None of the other proteoglycan populations from cartilage, tendon or aorta, even those similar in size and chemical structure, had this effect. Alkali treatment of the small proteoglycan of tendon eliminated its ability to inhibit fibrillogenesis, whereas chondroitinase digestion did not. This indicates that its interaction with collagen depends on the core protein. Fibrillogenesis of pepsin-digested collagens was affected similarly, indicating that interaction with the collagen telopeptides is not involved. The results suggest that interactions between collagen and proteoglycans may be quite specific both for the type of proteoglycan and its tissue of origin.     

Proteoglycans of fetal bovine tendon

The proteoglycans (PG) of bovine fetal tendon (4-8 months in utero) were extracted with 4 M guanidine HCl and partially purified by ion exchange chromatography. Proteoglycans from fetal tendon were virtually entirely small molecules (Kav approximately equal to 0.55 by Sepharose CL-4B chromatography). These small proteoglycans had dermatan sulfate glycosaminoglycan chains and a core protein (after chondroitinase ABC digestion) with Mr approximately equal to 45,000 on sodium dodecyl sulfate-polyacrylamide gels. By electrophoretic mobility, immunocross-reactivity, and V8 protease sensitivity, these proteoglycans were determined to be of both PG I and PG II types. In contrast, adult tendon contains only the PG II type of small proteoglycan. Proteoglycans synthesized by fetal tendon explant cultures were, by both chromatographic and electrophoretic mobilities, somewhat larger than those extracted from the same tissue. There was no difference in the spectrum of proteoglycans observed between those regions of fetal tendon destined to receive only tensional forces (proximal) and those regions that will be subjected as well to compressive forces (distal) in the adult. These observations indicate that the proteoglycan content and synthetic capability of all regions of fetal tendon are constant and significantly different from those of both the tensional and fibrocartilaginous regions of adult tendon.     

Isoforms of corneal keratan sulfate proteoglycan

Bovine corneal keratan sulfate proteoglycan was found to contain three major protein components. Two proteins (37 and 25 kDa) were released from the proteoglycan by endo-beta-galactosidase, N-glycanase, or chemical deglycosylation. A smaller protein (20 kDa), not covalently linked to keratan sulfate, co-purified with the proteoglycan by conventional and high performance ion exchange chromatography, by ethanol precipitation, and by affinity purification on columns of monoclonal antibody to keratan sulfate, but could be separated from the proteoglycan by gel filtration chromatography in dissociative agents. The three proteins produced different fragmentation patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after digestion with V8 protease, and each had unique two-dimensional tryptic peptide maps. The N-terminal amino acid sequence of the core proteins differed. In addition, the proteoglycans containing these proteins differed in molecular size, suggesting different levels of glycosylation of the two core proteins. Similarity of the core proteins was suggested by similar amino acid composition, similarities in tryptic maps, and antigenic cross-reactivity. Corneal keratan sulfate proteoglycan, therefore, seems to occur in two different, but related, forms whose core proteins may represent members of a homologous family.     

Characterisation of proteoglycans and their catabolic products in tendon and explant cultures of tendon.

Tendons are collagenous tissues made of mainly Type I collagen and it has been shown that the major proteoglycans of tendons are decorin and versican. Little is still known about the catabolism of these proteoglycans in tendon. Therefore, the aim of the study was to characterise the proteoglycans including their catabolic products present in uncultured bovine tendon and in the explant cultures of tendon. In this study, the proteoglycans were extracted from the tensile region of deep flexor tendon and isolated by ion-exchange chromatography and after deglycosylation analysed by SDS-polyacrylamide electrophoresis, Western blotting and amino-terminal amino acid sequence analysis. Based on amino acid sequence analysis, approximately 80% of the total proteoglycan core proteins in fresh tendon was decorin. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V(0) and/or V(1)) and aggrecan. Approximately 35% of decorin present in the matrix showed carboxyl-terminal proteolytic processing at a number of specific sites. The analysis of small proteoglycans lost to the medium of tendon explants showed the presence of biglycan and decorin with the intact core protein as well as decorin fragments that contained the amino terminus of the core protein. In addition, two core protein peptides of decorin starting at residues K(171) and D(180) were observed in the matrix and one core protein with an amino-terminal sequence commencing at G(189) was isolated from the culture medium. The majority of the large proteoglycans present in the matrix of tendon were degraded and did not contain the G1 globular domain. Furthermore the aggrecan catabolites present in fresh tendon and lost to the medium of explants were derived from aggrecanase cleavage of the core protein at residues E(373)-A(374), E(1480)-G(1481) and E(1771)-A(1772). The analysis of versican catabolites (splice variants V(0) and/or V(1)) also showed evidence of degradation of the core protein by aggrecanase within the GAG-beta subdomain, as well as cleavage by other proteinase(s) within the GAG-alpha and GAG-beta subdomains of versican (variants V(0) and/or V(2)). Degradation products from the amino terminal region of type XII collagen were also detected in the matrix and medium of tendon explants. This work suggests a prominent role for aggrecanase enzymes in the degradation of aggrecan and to a lesser extent versican. Other unidentified proteinases are also involved in the degradation of versican and small leucine-rich proteoglycans.     

Purification and analysis of the core protein of the protease-resistant intracellular chondroitin sulfate E proteoglycan from the interleukin 3-dependent mouse mast cell

Chondroitin sulfate E proteoglycan was extracted in the presence of protease inhibitors from 6 X 10(9) mouse bone marrow-derived, interleukin 3-dependent mast cells, of which 3 X 10(7) had been biosynthetically labeled with [35S]sulfate or [3H]glycine. Chondroitin sulfate E proteoglycan was purified to apparent homogeneity by density-gradient centrifugation, differential molecular weight dialysis, DEAE-52 ion exchange chromatography, and Sepharose CL-4B gel filtration chromatography. Chondroitin sulfate E proteoglycan, radiolabeled with [3H]glycine or [35S]sulfate, filtered as a single peak of radioactivity on Sepharose CL-4B with a Kav of 0.41. When purified [3H]glycine-labeled proteoglycan was digested with chondroitinase ABC and subjected to gel filtration, all of the radioactivity was shifted to a lower molecular weight. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis the Mr of the peptide core obtained by chondroitinase ABC treatment was approximately 10,000. The purified proteoglycan was resistant to degradation by collagenase, clostripain, trypsin, chymotrypsin, elastase, chymopapain, V8 protease, proteinase K, and Pronase, as assessed by gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the core peptide of the intact proteoglycan revealed that glycine, serine, and glutamic acid/glutamine accounted for 70% of the total amino acids and were present in a molar ratio of 4.3/1.6/1.0. When analyzed for neutral hexose content by gas-liquid chromatography, the proteoglycan contained approximately 2% of its weight as mannose, fucose, galactose, and other sugars, indicating that oligosaccharides were linked to the peptide core. The mouse bone marrow-derived mast cell chondroitin sulfate E proteoglycan, like the rat serosal mast cell heparin proteoglycan, is markedly protease resistant, has highly sulfated glycosaminoglycans, and contains a peptide core that is rich in serine and glycine. These characteristics of the mast cell class of intracellular proteoglycans may contribute to their function in stimulus-induced granule secretion as well as in mediator storage, including retention of cationic neutral proteases.     

The primary structure of link protein from rat chondrosarcoma proteoglycan aggregate

Cartilage proteoglycan monomers associate with hyaluronic acid to form proteoglycan aggregates. Link protein, a glycoprotein interacting with both hyaluronic acid and proteoglycan, serves to stabilize the aggregate structure. The primary structure of the link protein has been determined with a view to defining its interaction with both hyaluronic acid and proteoglycan. Thus, the link protein has been digested with staphylococcal V8 protease, trypsin, and chymotrypsin and the resulting peptides characterized by amino acid composition and sequence. We have determined that the link protein is a single peptide with 339 amino acid residues. The protein core has a molecular weight of 38,564. There is one N-linked oligosaccharide at residue 41 with a molecular weight of approximately 2,500. There are five disulfide bonds which define three loops within the amino acid sequence. The loop nearest to the NH2-terminal contains 78 amino acids and is followed by a section of 42 amino acids between it and the second loop. The second and third loops display considerable homology with each other; they consist of 71 and 70 amino acids, respectively, each contain two disulfide bonds, and both loops possess, approximately centrally, an epitope for the species nonspecific anti-link protein monoclonal antibody, 8A4. These loops are separated by a short section of 27 amino acids. We speculate that these loops are functionally important in the interaction of link protein with hyaluronic acid, as they appear to be the most conserved regions of link protein between species.     

Isolation and some properties of bovine brain 100 kDa heat shock protein

1. The 100 kDa protein was purified from bovine brains. 2. The antibody against the 100 kDa brain protein was prepared and was monospecific to the antigen. 3. The antibody cross-reacted with HeLa cell HSP100 (100 kDa heat shock protein). 4. The physicochemical, immunochemical properties and a partially amino acid sequence indicated that the 100 kDa protein was HSP100. 5. Peptide mapping using Staphylococcus aureus V8 protease showed a core peptide with 10 kDa molecular mass common to both HSP100 and HSP90. 6. The amino acid sequence of the 10 kDa fragment of the 100 kDa protein showed a high homology with that of human HSP90 (38-60); the difference was only two of 23 amino acid residues determined.     

Amino acid sequence of a peptide from keratan sulfate II-core protein linkage regions

Keratan sulfate II was prepared from the proteolytic digest of pig nucleus pulposus proteoglycan. The polysaccharide chains containing the fragment peptides of the core protein at their reducing terminal were subjected to anhydrous HF-solvolysis reaction and one of the glycopeptides from the keratan sulfate II-core protein linkage regions was isolated. The amino acid sequence of the peptide was deduced to be Ala-Pro-Ser-Pro-Gly, which is different from those reported for the attachment sites of chondroitin sulfate on core proteins from various sources. The results provided the first solid amino acid sequence for the keratan sulfate II-core protein linkage regions and suggested that the amino acid sequence of the core protein might determine the distribution of chondroitin sulfates and keratan sulfates along the core protein of the proteoglycan molecule.     

Characterization of proteoglycans from adult bovine tendon

Proteoglycans were extracted in good yield from the proximal, fibrous portion of adult bovine tendon with 4 m guanidine HCl. They comprise less than 1% of the dry weight of the tissue. Using CsCl density gradient centrifugation, gel chromatography, and ion exchange chromatography, two populations of proteoglycans were separated and purified from other tissue proteins. One was a large, chondroitin sulfate proteoglycan with high buoyant density in CsCl. This component appeared to be composed of two or three subpopulations as detected by agarose/polyacrylamide electrophoresis, although they could not be effectively separated from one another for individual characterization. As a group, the large proteoglycans eluted from Sepharose CL-2B with Kav from 0.1-0.5 and their core protein had Mr greater than 200,000 with high contents of glutamic acid, serine, and glycine. The glycosaminoglycan chains had a weight average Mr of 17,000 and more than 98% of the uronic acid was glucuronic acid. This group comprised only 12% of the total proteoglycan of the tissue. The other 88% of the proteoglycans appeared to represent one group of small molecules that eluted from Sepharose CL-2B at Kav = 0.70. They demonstrated buoyant densities in a CsCl gradient ranging from greater than or equal to 1.51 to 1.30 g/ml. Their core protein had an apparent Mr = 48,000 following removal of the glycosaminoglycan chains by digestion with chondroitinase ABC. This core protein had a particularly high content of aspartic acid/asparagine and leucine. The glycosaminoglycan chains had a weight average Mr of 37,000 and were dermatan sulfate containing 73% iduronic acid. Those molecules found at highest buoyant density appeared to have additional glycosaminoglycan chains that were shorter. Proteoglycans were also extracted from the pressure-bearing distal region of this tendon, where contents of proteoglycan per wet weight of tissue were 3-fold higher and as much as 50% of this was as large as the large proteoglycans from the proximal tissue. Preparations of large proteoglycans from both tendon regions contained molecules capable of interacting with hyaluronic acid.     

Interaction of decorin with CNBr peptides from collagens I and II. Evidence for multiple binding sites and essential lysyl residues in collagen.

Decorin is a small leucine-rich chondroitin/dermatan sulfate proteoglycan reported to interact with fibrillar collagens through its protein core and to localize at d and e bands of the collagen fibril banding pattern. Using a solid-phase assay, we have determined the interaction of peptides derived by CNBr cleavage of type I and type II collagen with decorin extracted from bovine tendon and its protein core and with a recombinant decorin preparation. At least five peptides have been found to interact with all three decorin samples. The interaction of peptides with tendon decorin has a dissociation constant in the nanomolar range. The triple helical conformation of the peptide trimeric species is a necessary requisite for the binding. All positive peptides have a region within the d and e bands of collagen fibrils. Two chemical derivatives of collagens and of positive peptides were prepared by N-acetylation and N-methylation of the primary amino group of Lys/Hyl side chains. Chemical modifications performed in mild conditions do not significantly alter the thermal stability of peptide trimeric species whereas they affect the interaction with decorin: N-acetylation eliminates both the positive charge and the binding to decorin, whereas N-methylation preserves the cationic character and modulates the binding. We conclude that decorin makes contacts with multiple sites in type I collagen and probably also in type II collagen and that some collagen Lys/Hyl residues are essential for the binding.     

The N-terminal sequence of the large proteoglycan of articular cartilage

A peptide with hyaluronic acid-binding properties was isolated from trypsin digests of bovine articular cartilage proteoglycan aggregate. This peptide originated from the N-terminus of the proteoglycan core protein, retained its function of forming complexes with hyaluronate and link protein and contained at least one keratan sulfate chain. Amino acid sequence data demonstrated that the first six amino acid residues of the N-terminus of bovine articular cartilage proteoglycan core protein differed from the same region from the rat chondrosarcoma proteoglycan. Further sequence data indicate areas of considerable sequence homology in the hyaluronic acid-binding regions of proteoglycans from the two species.     

Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma

We have studied the extractability of type IV collagen, laminin, and heparan sulfate proteoglycan from EHS tumor tissue growth in normal and lathyritic animals. Laminin and heparan sulfate proteoglycan were readily extracted with chaotropic solvents from both normal and lathyritic tissue. The collagenous component was only solubilized from lathyritic tissue in the presence of a reducing agent. These results indicate that lysine-derived cross-links and disulfide bonds stabilize the collagenous component in the matrix but not the laminin or the heparan sulfate proteoglycan. The majority of the collagen present in the extracts had a native triple helix based upon the pattern of peptides resistant to pepsin digestion and visualization in the electron microscope by the rotary shadow technique. This protein was composed of chains (Mr 185000 and 170000) identical in migration to the chains of newly synthesized type IV procollagen. This finding confirms earlier work that indicates that the biosynthetic form, type IV procollagen, is incorporated as such in the basement membrane matrix. Material with smaller chains (Mr 160000 and 140000) appeared on storage in acetic acid solutions. These results indicate that the lower molecular weight collagen in acid extracts of basement membrane arises artifactually due to an endogenous acid-active protease.     

Isolation of a cDNA that encodes the peptide core of the secretory granule proteoglycan of rat basophilic leukemia-1 cells and assessment of its homology to the human analogue

It has been previously shown that a single gene is used to encode the peptide core of the extracellular proteoglycan of rat L2 yolk sac tumor cells and the intracellular proteoglycan of rat basophilic leukemia (RBL)-1 cells. In order to determine if the predicted amino acid sequences of these proteoglycans are identical as well as to isolate a full length cDNA encoding a rat secretory granule proteoglycan, a cDNA library was prepared from RBL-1 cells and screened with the 165-base pair 5'----XmnI fragment of pPG-1, a partial cDNA which encodes the rat L2 cell proteoglycan peptide core. Based on the consensus nucleotide sequence of two full length RBL-1 cell-derived cDNAs, the 5' untranslated region of the mRNA that is expressed in RBL-1 cells is shorter than that expressed in the rat L2 cells although the coding regions of the mRNAs from the two cell types are identical. These findings indicate that the targeting of proteoglycans to an intracellular or extracellular compartment is a cell-specific event which is independent of the translated peptide core. Since the RBL-1 cell and the rat L2 cell proteoglycans have different types of glycosaminoglycans bound to them, it can also be concluded that the selection of the type of glycosaminoglycan that will be synthesized onto a peptide core is a cell-specific event which is not exclusively dependent on the translated peptide core. When the predicted amino acid sequence of the RBL-1 cell proteoglycan peptide core was compared to the predicted sequence of the homologous human molecule from HL-60 cells, 48% of the amino acids were identical. The N terminus was the most highly conserved area of the molecule. This region of the peptide core, which precedes the serine-glycine repeat region, is likely to be of critical importance for the biosynthesis and/or function of these proteoglycans. Analysis of 10 different mouse/hamster somatic cell hybrid lines with a SspI----3' fragment of the rat L2 cell cDNA revealed that, as in the human, the gene that encodes the mouse analogue of this peptide core resides on chromosome 10.     

Structural organization of the lens fiber cell plasma membrane protein MP18

The 18,000-dalton bovine lens fiber cell intrinsic membrane protein MP18 was phosphorylated on a serine residue by both cAMP-dependent protein kinase and protein kinase C. In addition, this protein bound calmodulin and was recognized by a monoclonal antibody (2D10). These different regions were localized using enzymatic and chemical fragmentation of electrophoretically purified MP18 that had been phosphorylated with either cAMP-dependent protein kinase or protein kinase C. Partial digestion of 32P-labeled MP18 with protease V8 resulted in a Mr = 17,000 peptide that bound calmodulin, but neither contained 32P or was recognized by the monoclonal antibody 2D10. Furthermore, the 17-kDa peptide had the same N-terminal amino acid sequence as MP18. Thus, the monoclonal antibody 2D10 recognition site and the protein kinase phosphorylation site(s) are close together and confined to a small region in the C terminus of MP18. This conclusion was confirmed in experiments where MP18 was fragmented with trypsin, endoproteinase Lys-C, or CNBr. The location of the phosphorylation site was confirmed by sequencing the small 32P-labeled, C-terminal peptide that resulted from protease V8 digestion of 32P-labeled MP18. This peptide contained a consensus sequence for cAMP-dependent protein kinase.     

Proteoglycan synthesis by fibroblast cultures initiated from regions of adult bovine tendon subjected to different mechanical forces

Fibroblast cultures were initiated from two distinct regions of the adult bovine deep flexor tendon and synthesis of 35S-labeled proteoglycans by these cultures was investigated. The proximal/tensional region of the tendon was composed of linearly arranged dense collagen bundles, and its glycosaminoglycan hexosamine content was only 0.2% of the dry weight of the tissue. The proteoglycans of this region were predominantly small (Kav = 0.5 on Sepharose CL-4B). Cells placed into culture from this region attached to the substratum readily, and the radiolabeled proteoglycans from these cultures were 90% small proteoglycans. In a more distal region of the tendon that is subjected to compressive forces, the collagen was arranged as a network of fibrils separated from each other by a matrix that stained intensely with Alcian blue. The glycosaminoglycan content of this compressed region was up to 5-fold higher than in the proximal region, and as much as 50% of the proteoglycans were large molecules (eluted from Sepharose CL-4B in the Vo). Cells placed into culture from the distal/compressed region did not attach to the substratum as readily as those from the proximal region and were characterized by the presence of numerous cytoplasmic lipid inclusions. The [35S]proteoglycans synthesized by the distal tendon fibroblast cultures were divided into two approximately equal populations of large and small proteoglycans having elution characteristics similar to the proteoglycans extracted from this tissue. The distinct profiles of proteoglycan production were maintained by the cells in culture for several weeks, although eventually the amount of large proteoglycan synthesized by the distal tendon fibroblast cultures diminished. Both regions of tendon contained predominantly type I collagen, and collagen production was about 10% of the total protein synthesized by both cell cultures. These observations indicate that adult tendon fibroblasts in culture express stable synthesis of proteoglycan populations similar to those found in the region of tendon from which they were derived.     

The proteoglycan content and the axial periodicity of collagen in tendon.

The glycosaminoglycan content and the axial periodicity of collagen was determined in various regions of the rabbit flexor digitorum profundus tendon. This tendon, which passes from the calf to the toes round the inner side of the ankle, contains a thickened sesamoid-like pad where it is subjected to friction and pressure. Other regions of the tendon are subject only to longitudinal tension. In tensional areas the axial periodicity of collagen was of the order of 62 nm and the tissue contained less than 0.2% proteoglycan on a dry weight basis. In the sesamoid-like region, however, the axial periodicity was a significant 13-15% less, and the proteoglycan constituted about 3.5% of the dry weight. Also, in the tensional areas the predominant glycosaminoglycan was dermatan sulphate, whereas in the sesamoid the predominant glycosaminoglycan was chondroitin sulphate. The possible interrelationships between collagen axial peroidicity and proteoglycan content in this tissue are discussed.     

Chick cartilage chondroitin sulfate proteoglycan core protein. II. Nucleotide sequence of cDNA clone and localization of the S103L epitope.

A lambda gt11 expression library containing cDNA from total chick embryo was screened with S103L, a rat monoclonal antibody which reacts specifically with the core protein of the chick cartilage chondroitin sulfate proteoglycan. One clone was identified which produced a 220-kDa beta-galactosidase/S103L-binding fusion protein. Sequencing the entire 1.5-kilobase cDNA insert showed that it contained a single open reading frame, which encoded a portion of the proteoglycan core protein from the chondroitin sulfate domain. This was confirmed by comparison with amino acid sequence data from peptide CS-B, which was derived from the chondroitin sulfate domain (Krueger, R.C., Jr., Fields, T. A., Hildreth, J., IV, and Schwartz, N.B. (1990) J. Biol. Chem. 265, 12075-12087). Furthermore, the 3' end of the insert overlapped with 23 bases at the 5' end of the published sequence for the C-terminal globular domain (Sai, S., Tanaka, T., Kosher, R. A., and Tanzer, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5081-5085), which oriented this clone, as well as the CS peptide, along the protein core. The cDNA insert hybridized with a 9-kilobase mRNA from sternal chondrocytes as well as a similar sized message in brain but did not hybridize to any message from rat chondrosarcoma or from undifferentiated limb bud mesenchyme. In further studies, the fusion protein as well as a cyanogen bromide fragment (70 kDa) derived from it were isolated and shown to react with S103L, indicating that cleavage at methionine residues does not disrupt the antibody recognition site. Purification and N-terminal sequencing of the antigenic CNBr fragment derived from the fusion protein revealed that its N terminus is preceded by a methionine in the fusion protein and overlaps with the N terminus of peptide CS-B. As peptide CS-B is not recognized by S103L and the C terminus of peptide CS-B lies beyond the proteoglycan portion of the antigenic CNBr fragment, the S103L epitope is either contained within the 11 amino acids preceding the N terminus of peptide CS-B or it spans the clostripain cleavage site at the origin of the N terminus of peptide CS-B.