This review focuses on the extracellular proteoglycans. Special emphasis is placed on the structural features of their protein cores, their gene organization, and their transcriptional control. A simplified nomenclature comprising two broad groups of extracellular proteoglycans is offered: the small leucine-rich proteoglycans or SLRPs, pronounced "slurps, " and the modular proteoglycans. The first group encompasses at least five distinct members of a gene family characterized by a central domain composed of leucine-rich repeats flanked by two cysteine-rich regions. The second group consists of those proteoglycans whose unifying feature is the assembly of various protein modules in a relatively elongated and often highly glycosylated structure. This group is quite heterogeneous and includes a distinct family of proteoglycans, the "hyalectans," that bind hyaluronan and contain a C-type lectin motif that is likely to bind carbohydrates, and a less distinct group that contains structural homologies but lacks hyaluronan-binding properties or lectin-like domains.

Perlecan is a modular heparan sulfate proteoglycan that is localized to cell surfaces and within basement membranes. Its ability to interact with basic fibroblast growth factor (bFGF) suggests a central role in angiogenesis during development, wound healing, and tumor invasion. In the present study we investigated, using domain specific anti-perlecan monoclonal antibodies, the binding site of bFGF on human endothelial perlecan and its cleavage by proteolytic and glycolytic enzymes. The heparan sulfate was removed from perlecan by heparitinase treatment, and the ϳ450-kDa protein core was digested with various proteases. Plasmin digestion resulted in a large fragment of ϳ300 kDa, whereas stromelysin and rat collagenase cleaved the protein core into smaller fragments. All three proteases removed immunoreactivity toward the anti-domain I antibody. We showed also that perlecan bound bFGF specifically by the heparan sulfate chains located on the amino-terminal domain I. Once bound, the growth factor was released very efficiently by stromelysin, rat collagenase, plasmin, heparitinase I, platelet extract, and heparin. Interestingly, heparinase I, an enzyme with a substrate specificity for regions of heparan sulfate similar to those that bind bFGF, released only small amounts of bFGF. Our findings provide direct evidence that bFGF binds to heparan sulfate sequences attached to domain I and support the hypothesis that perlecan represents a major storage site for this growth factor in the blood vessel wall. Moreover, the concerted action of proteases that degrade the protein core and heparanases that remove the heparan sulfate may modulate the bioavailability of the growth factor.

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Human bone marrow stem cells (hMSCs) have been shown to differentiate in vitro into a number of cell lineages and are a potential autologous cell source for the repair and replacement of damaged and diseased musculoskeletal tissues. hMSC differentiation into chondrocytes has been described in highdensity cell pellets cultured with specific growth and differentiation factors. We now describe how culture of hMSCs as a shallow multicellular layer on a permeable membrane over 2-4 weeks resulted in a much more efficient formation of cartilaginous tissue than in established chondrogenic assays. In this format, the hMSCs differentiated in 14 days to produce translucent, flexible discs, 6 mm in diameter by 0.8 -1 mm in thickness from 0.5 ؋ 10 6 cells. The discs contained an extensive cartilage-like extracellular matrix (ECM), with more than 50% greater proteoglycan content per cell than control hMSCs differentiated in standard cell pellet cultures. The disc constructs were also enriched in the cartilage-specific collagen II, and this was more homogeneously distributed than in cell pellet cultures. The expression of cartilage matrix genes for collagen type II and aggrecan was enhanced in disc cultures, but improved matrix production was not accompanied by increased expression of the transcription factors SOX9, L-SOX5, and SOX6. The fast continuous growth of cartilage ECM in these cultures up to 4 weeks appeared to result from the geometry of the construct and the efficient delivery of nutrients to the cells. Scaffold-free growth of cartilage in this format will provide a valuable experimental system for both experimental and potential clinical studies.

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Perlecan, a multidomain heparan sulfate proteoglycan (PG), is an intrinsic component of basement membranes and extracellular matrices. We used a prokaryotic expression vector to generate fusion proteins encoding various domains of human perlecan protein core and these recombinant proteins were used as immunogens to produce mouse anti-human monoclonal antibodies (MAb). One MAb, designated 7B5, was characterized by Western blotting and ELISA and was shown to react specifically with the laminin-like region of perlecan (Domain III) but not with two other fusion proteins encoding Domain II or V. This perlecan epitope was detected by immunoenzymatic staining in the basement membranes of human tissues including pituitary gland, skin, breast, thymus, prostate, colon, liver, pancreas, spleen, heart, and lung. All vascular basement membranes tested contained this gene product. In addition, sinusoidal vessels of liver, spleen, lymph nodes, and pituitary gland expressed high levels of perlecan in the subendothelial region. In situ hybridization, using as probe the same human cDNA-encoding Domain III, localized perlecan mRNA to specific cell types within the tissues and demonstrated that in skin, perlecan appears to be synthesized exclusively by connective tissue cells in the dermal layer. The availability of MAb against precise regions of human perlecan will allow the investigation of this gene product in normal and diseased states.

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The proteoglycans aggrecan, versican, neurocan, and brevican bind hyaluronan through their N-terminal G1 domains, and other extracellular matrix proteins through the C-type lectin repeat in their C-terminal G3 domains. Here we identify tenascin-C as a ligand for the lectins of all these proteoglycans and map the binding site on the tenascin molecule to fibronectin type III repeats, which corresponds to the proteoglycan lectinbinding site on tenascin-R. In the G3 domain, the C-type lectin is flanked by epidermal growth factor (EGF) repeats and a complement regulatory protein-like motif. In aggrecan, these are subject to alternative splicing. To investigate if these flanking modules affect the C-type lectin ligand interactions, we produced recombinant proteins corresponding to aggrecan G3 splice variants. The G3 variant proteins containing the C-type lectin showed different affinities for various ligands, including tenascin-C, tenascin-R, fibulin-1, and fibulin-2. The presence of an EGF motif enhanced the affinity of interaction, and in particular the splice variant containing both EGF motifs had significantly higher affinity for ligands, such as tenascin-R and fibulin-2. The mRNA for this splice variant was shown by reverse transcriptase-PCR to be expressed in human chondrocytes. Our findings suggest that alternative splicing in the aggrecan G3 domain may be a mechanism for modulating interactions and extracellular matrix assembly.The aggregating proteoglycans aggrecan, versican, neurocan, and brevican form the lectican (1) or hyalectan (2) family and are major components of the extracellular matrix (ECM) 1 with important functions in many tissues. The core proteins of these proteoglycans have extended central glycosaminoglycan attachment regions of varying length that are flanked by globular domains (3-6). In the cartilage proteoglycan aggrecan, the large extent of glycosaminoglycan side chain substitution and the resulting fixed charge density attracts counter-ions and water through osmotic processes. The resulting swelling pressure is crucial for the biomechanical properties of this tissue (7). The conserved N-terminal globular G1 domains anchor these proteoglycans to hyaluronan in an interaction stabilized by the link protein (8 -12). Aggrecan contains an additional globular G2 domain of unknown function between the G1 domain and the glycosaminoglycan attachment region (13). The C-terminal G3 domain is highly conserved and found in all four of these proteoglycans.We have shown previously that the G3 domain mediates binding to other ECM molecules, e.g. tenascin-R (14, 15), fibulin-1 (16), fibulin-2 (17), and fibrillin-1 (18). The G3 domain also binds sulfated glycolipids on the cell surface (19). In addition, neurocan has been reported to bind to tenascin-C (20). The ECM protein ligands for the G3 domains are all dimeric or multimeric proteins, and we have shown that they can crosslink proteoglycans from different hyaluronan/proteoglycan aggregates (17). This may well be of functional importance for the organi...