Molecular polymorphism of the syndecans. Identification of a hypo-glycanated murine syndecan-1 splice variant.

We have identified a cDNA that encodes a variant form of murine syndecan-1. The variant cDNA lacks the sequence corresponding to the first 132 nucleotides of the third exon of the syndecan-1 gene. The corresponding message is rare. The alternative splice respects the reading frame and deletes 44 amino acids from the protein, joining the S45GS47GT sequence to a variant immediate downstream context. This sequence context initiates with alanine instead of glycine as residue 50, reducing the number of SGXG sequence motifs in the protein from two to one. Expression of this variant syndecan-1 in Madin-Darby canine kidney or MOLT-4 cells yielded a recombinant proteoglycan with a reduced number and clustering of the heparan sulfate chains. Both the conversions of Ala50 and of Lys53 into glycine enhanced the heparan sulfate substitution of the variant protein. These findings support the concept that serine-glycine dipeptide signals for glycosaminoglycan/heparan sulfate synthesis depend on sequence context (Zhang, L., David, G., and Esko, J. D. (1995) J. Biol. Chem. 270, 27127-27135) and imply that alternative splicing mechanisms may in part control the molecular polymorphism of syndecan-1 and, therefore, the efficiency and versatility of this protein in its co-receptor functions.

Changes in glycosaminoglycan structure and composition of the main heparan sulphate proteoglycan from human colon carcinoma cells (perlecan) during cell differentiation.

Colon carcinoma cells provide a useful model to study the biochemical processes associated with cell differentiation. Undifferentiated HT29, differentiated HT29MTX(-3) and HT29MTX(-6), and Caco2 human colon carcinoma cells have been used to study the production of proteoglycans and to characterize the glycosaminoglycan structure of the heparan sulphate chains. All the cell lines produce mainly a heparan sulphate proteoglycan that is found partly in the extracellular medium and associated to the cell membrane. The heparan sulphate proteoglycans from the media were purified by ion-exchange chromatography and subjected to structural analysis. The heparan sulphate proteoglycan from differentiated cells is larger and more homogeneous in size than the heparan sulphate proteoglycan from undifferentiated HT29 cells. No differences in protein core structure were observed when cells were labeled with [35S]methionine and the protein cores visualized by gel electrophoresis. Nevertheless, differences in glycosaminoglycan composition were found correlated with the degree of differentiation. The heparan sulphate chains from differentiated HT29MTX(-3) and HT29MTX(-6) cells have a higher sulphation degree than those from undifferentiated HT29 cells. The heparan sulphate from Caco2 cells is the most highly sulphated species. The differences are mainly attributed to O-sulphate groups. The increase in O-sulphation was more pronounced for D-glucosamine 6-O-sulphate than for L-iduronic acid 2-O-sulphate groups.

Enterocytic differentiation correlates with changes in the fine structure and sulfation of perlecan in HT29 human colon carcinoma cells.

Undifferentiated HT29 and differentiated HT29G-human colon carcinoma cells have been used to study the changes in proteoglycan production and structure associated with enterocytic cell differentiation. Differentiated cells incorporate twice as much sulfate than undifferentiated cells when labeled with [35S]sulfate. Both cell lines produce a heparan sulfate proteoglycan which was purified by ion-exchange. The heparan sulfate proteoglycan from differentiated HT29G- cells is larger and more homogeneous in size than that produced by undifferentiated HT29 cells. No differences in the core protein structure were observed. The detailed structural analysis of the heparan sulfate chains revealed that the structure of these chains follows the standard rules for these glycosaminoglycans with N-sulfated domains and N-acetylated domains. The main finding was that differentiated HT29G- cells have a degree of higher sulfation than HT29 cells. These differences were found to affect primarily 6-O-sulfated positions.

The effect of TGF-beta 1 on cell proliferation and proteoglycan production in human melanoma cells depends on the degree of cell differentiation.

The effect of transforming growth factor beta 1 (TGF-beta 1) on cell proliferation, colony formation in soft agar and synthesis and structure of proteoglycans was studied in three human melanoma cell lines at different stages of differentiation: SK-mel-1.36-1-5 (early), SK-mel-3.44 (intermediate) and SK-mel-23 (late). TGF-beta 1 potently inhibited cell growth in monolayer as well as in soft agar. TGF-beta 1 increased the release of sulfated proteoglycans into the medium, including the cell-specific melanoma proteoglycan, mel-PG, and induced changes in disaccharide composition and sulfation of the glycosaminoglycan chains. In all the cases, the effect of TGF-beta 1 was more pronounced in the most undifferentiated cell line SK-mel-1.36-1-5 than in the SK-mel-3.44, whereas it had no effect on the most differentiated SK-mel-23 cells.

Transforming growth factor beta 1 increases the synthesis and shedding of the melanoma-specific proteoglycan in human melanoma cells.

Melanoma cells produce a cell-specific proteoglycan, mel-PG, which is an integral chondroitin sulfate proteoglycan that can be released into the medium as a result of the proteolytic cleavage of the trans-membrane form. The effect of transforming growth factor beta 1 (TGF-beta 1) on proteoglycan production was studied in three melanoma cell lines at various stages of differentiation: SK-mel-1.36-1-5 (early), SK-mel-3.44 (intermediate), and SK-mel-23 (late). The main effect of TGF-beta 1 was to increase the synthesis and shedding of mel-PG into the medium without affecting the amount present in the cell membranes nor the balance between the proteoglycan and the glycoprotein forms of mel-PG. After TGF-beta 1 treatment, there was an increase in the amount of mel-PG present in the medium as observed in metabolic labeling, immunoprecipitation, and pulse-chase experiments. This effect was more pronounced in the SK-mel-1.36-1-5 than in the SK-mel-3.44 cell line, whereas the SK-mel-23 cells did not contain mel-PG either in the presence or in the absence of TGF-beta 1. Characterization of mel-PG purified from the medium from control and TGF-beta 1-treated cells showed that the factor increased slightly the GAG chain length in SK-mel-1.36-1-5 but not in SK-mel-3.44 cells, without modifying the degree of sulfation.

Heparan sulfate proteoglycans. Essential co-factors in receptor-mediated processes with relevance to the biology of the vascular wall.

Heparan sulfate (HS), a mixed bag of complex, heterogeneous and highly charged polysaccharides, is an essential co-factor in a large number of receptor-ligand interactions and cellular pathways. These co-factor functions depend on the binding-interactions of the HS chains with the ligand or receptor, or both. These binding interactions and the ensuing functional effects often depend on defined carbohydrate sequences within the HS chains, whereby the required sequences are not always represented within all natural forms of the polysaccharide. The proteins that are substituted with HS resort from a limited number of protein families, with different cellular, subcellular and supramolecular associations, and show differential activities in functional assays. It is likely that the natural co-factor functions of the HS proteoglycans depend on glycan-protein and protein-protein interactions that are subject to modulation, both at the glycan and protein levels.

Effect of transforming growth factor-beta 1 and basic fibroblast growth factor on the expression of cell surface proteoglycans in human lung fibroblasts. Enhanced glycanation and fibronectin-binding of CD44 proteoglycan, and down-regulation of glypican.

We have tested the effects of transforming growth factor-beta 1 (TGF-beta 1), basic fibroblast growth factor (bFGF) and TGF-beta 1 + bFGF on the expression of the cell surface proteoglycans (CD44, syndecans and glypican) in cultures of human lung fibroblasts (HLF). Cell surface proteoglycan expression was monitored by quantitative immunoprecipitation from metabolically labelled cells. Western and Northern blotting and evaluation of the glycanation of the proteoglycans. Stimulation of the cells with TGF-beta 1 increased the length of the chondroitin sulphate (CS) chains on CD44 (approximately 1.6-fold). bFGF, administered solely, also increased the length of the CS chains on CD44 (approximately 1.4-fold), whereas the combination of TGF-beta 1 + bFGF nearly doubled both the length and the number of the CS chains on CD44. None of these treatments lead to changes in CD44 message or core-protein expression. This enhanced glycanation of CD44 after the TGF-beta 1, bFGF and combined treatments correlated with a 2-fold increase in the affinity of the proteoglycan for fibronectin but had no influence on the binding to type I collagen. TGF-beta 1, alone or in combination with bFGF, also stimulated the CS content of syndecan-1, but none of the other syndecans was significantly affected by any of the factors or combinations tested. The expression of glypican however was significantly decreased (nearly halved) by the combination of TGF-beta 1 + bFGF, less so by TGF-beta 1 and not at all by bFGF. This decrease occurred both at the level of the message and of the core protein. These data demonstrate specific and differential effects of TGF-beta 1 and bFGF on the structure, expression and interactions of the cell surface proteoglycans of HLF.

Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta.

We have analysed the interactions of three proteoglycans of the decorin family, decorin, biglycan and fibromodulin, with transforming growth factor beta (TGF-beta). The proteoglycan core proteins, expressed from human cDNAs as fusion proteins with Escherichia coli maltose-binding protein, each bound TGF-beta 1. They showed only negligible binding to several other growth factors. Intact decorin, biglycan and fibromodulin isolated from bovine tissues competed with the fusion proteins for the TGF-beta binding. Affinity measurements suggest a two-site binding model with Kd values ranging from 1 to 20 nM for a high-affinity binding site and 50 to 200 nM for the lower-affinity binding site. The stoichiometry indicated that the high-affinity binding site was present in one of ten proteoglycan core molecules and that each molecule contained a low-affinity binding site. Tissue-derived biglycan and decorin were less effective competitors for TGF-beta binding than fibromodulin or the non-glycosylated fusion proteins; removal of the chondroitin/dermatan sulphate chains of decorin and biglycan (fibromodulin is a keratan sulphate proteoglycan) increased the activities of decorin and biglycan, suggesting that the glycosaminoglycan chains may hinder the interaction of the core proteins with TGF-beta. The fusion proteins competed for the binding of radiolabelled TGF-beta to Mv 1 Lu cells and endothelial cells. Affinity labelling showed that the binding of TGF-beta to betaglycan and the type-I receptors in Mv 1 Lu cells and to endoglin in endothelial cells was reduced, but the binding to the type-II receptors was unaffected. TGF-beta 2 and 3 also bound to all three fusion proteins. Latent recombinant TGF-beta 1 precursor bound slightly to fibromodulin and not at all to decorin and biglycan. The results show that the three decorin-type proteoglycans each bind TGF-beta isoforms and that slight differences exist in their binding properties. They may regulate TGF-beta activities by sequestering TGF-beta into extracellular matrix.

Ras transformation alters the composition of extracellular matrix proteoglycans in rat fibroblasts.

ts371 KiMuSV-transformed rat kidney fibroblasts growing at the permissive temperature to allow the expression of a transforming p21 ras protein secrete a different pattern of chondroitin/dermatan sulfate proteoglycans than when growing at the non-permissive temperature. Transformed cells produce less versican and larger amounts of biglycan and decorin than their normal counterpart, and they do not secrete 35S-labeled PG-100. Western blot experiments showed the presence of PG-100 epitope, suggesting that the apparent effect of the oncoprotein is due to a reduced sulfation of the molecule. Transformed and non-transformed cells are similarly sensitive to the proteoglycan-induction effect of TGF-beta.

Differential effect of transforming growth factor beta on proteoglycan synthesis in human embryonic lung fibroblasts.

The effect of transforming growth factor beta (TGF-beta) on the biosynthesis of individual proteoglycans (PGs) by human embryonic lung fibroblasts has been investigated using specific antibodies and cDNA probes. Human lung fibroblasts secrete the two small chondroitin/dermatan sulfate PGs, PG-I or biglycan (300 kDa) and, in a larger proportion, PG-II or decorin (130 kDa). Metabolic labeling experiments reveal that TGF-beta induces selectively the expression of PG-I, whereas the level of PG-II remains unaltered. The effect of TGF-beta on PG-I and PG-II has been studied by immunoprecipitation and Northern blot analysis. Either at the core protein or mRNA level, a specific 5-fold increase in PG-I can be observed. TGF-beta acts probably at the transcriptional level, as actinomycin D blocks completely the TGF-beta induced proteoglycan synthesis. A low saturation density and a slower growth rate is also observed for TGF-beta treated cells. The possible role of PG-I and PG-II as mediators of the growth inhibition caused by TGF-beta is discussed.