WARP is a novel multimeric component of the chondrocyte pericellular matrix that interacts with perlecan

WARP is a novel member of the von Willebrand factor A domain superfamily of extracellular matrix proteins that is expressed by chondrocytes. WARP is restricted to the presumptive articular cartilage zone prior to joint cavitation and to the articular cartilage and fibrocartilaginous elements in the joint, spine, and sternum during mouse embryonic development. In mature articular cartilage, WARP is highly specific for the chondrocyte pericellular microenvironment and co-localizes with perlecan, a prominent component of the chondrocyte pericellular region. WARP is present in the guanidine-soluble fraction of cartilage matrix extracts as a disulfide-bonded multimer, indicating that WARP is a strongly interacting component of the cartilage matrix. To investigate how WARP is integrated with the pericellular environment, we studied WARP binding to mouse perlecan using solid phase and surface plasmon resonance analysis. WARP interacts with domain III-2 of the perlecan core protein and the heparan sulfate chains of the perlecan domain I with K(D) values in the low nanomolar range. We conclude that WARP forms macromolecular structures that interact with perlecan to contribute to the assembly and/or maintenance of "permanent" cartilage structures during development and in mature cartilages.     

A biomechanical role for perlecan in the pericellular matrix of articular cartilage

Chondrocytes are surrounded by a narrow pericellular matrix (PCM) that is biochemically, structurally, and biomechanically distinct from the bulk extracellular matrix (ECM) of articular cartilage. While the PCM is often defined by the presence of type VI collagen, other macromolecules such as perlecan, a heparan sulfate (HS) proteoglycan, are also exclusively localized to the PCM in normal cartilage and likely contribute to PCM structural integrity and biomechanical properties. Though perlecan is essential for normal cartilage development, its exact role in the PCM is unknown. The objective of this study was to determine the biomechanical role of perlecan in the articular cartilage PCM in situ and its potential as a defining factor of the PCM. To this end, atomic force microscopy (AFM) stiffness mapping was combined with dual immunofluorescence labeling of cryosectioned porcine cartilage samples for type VI collagen and perlecan. While there was no difference in overall PCM mechanical properties between type VI collagen- and perlecan-based definitions of the PCM, within the PCM, interior regions containing both type VI collagen and perlecan exhibited lower elastic moduli than more peripheral regions rich in type VI collagen alone. Enzymatic removal of HS chains from perlecan with heparinase III increased PCM elastic moduli both overall and locally in interior regions rich in both perlecan and type VI collagen. Heparinase III digestion had no effect on ECM elastic moduli. Our findings provide new evidence for perlecan as a defining factor in both the biochemical and biomechanical properties of the PCM.     

Perlecan maintains the integrity of cartilage and some basement membranes

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.     

Structural and functional mutations of the perlecan gene cause Schwartz-Jampel syndrome, with myotonic myopathy and chondrodysplasia

Perlecan, a large heparan sulfate proteoglycan, is a component of the basement membrane and other extracellular matrices and has been implicated in multiple biological functions. Mutations in the perlecan gene (HSPG2) cause two classes of skeletal disorders: the relatively mild Schwartz-Jampel syndrome (SJS) and severe neonatal lethal dyssegmental dysplasia, Silverman-Handmaker type (DDSH). SJS is an autosomal recessive skeletal dysplasia characterized by varying degrees of myotonia and chondrodysplasia, and patients with SJS survive. The molecular mechanism underlying the chondrodystrophic myotonia phenotype of SJS is unknown. In the present report, we identify five different mutations that resulted in various forms of perlecan in three unrelated patients with SJS. Heterozygous mutations in two patients with SJS either produced truncated perlecan that lacked domain V or significantly reduced levels of wild-type perlecan. The third patient had a homozygous 7-kb deletion that resulted in reduced amounts of nearly full-length perlecan. Unlike DDSH, the SJS mutations result in different forms of perlecan in reduced levels that are secreted to the extracellular matrix and are likely partially functional. These findings suggest that perlecan has an important role in neuromuscular function and cartilage formation, and they define the molecular basis involved in the difference in the phenotypic severity between DDSH and SJS.     

The leucine-rich repeat protein PRELP binds perlecan and collagens and may function as a basement membrane anchor

PRELP (proline arginine-rich end leucine-rich repeat protein) is a heparin-binding leucine-rich repeat protein in connective tissue extracellular matrix. In search of natural ligands and biological functions of this molecule, we found that PRELP binds the basement membrane heparan sulfate proteoglycan perlecan. Also, recombinant perlecan domains I and V carrying heparan sulfate bound PRELP, whereas other domains without glycosaminoglycan substitution did not. Heparin, but not chondroitin sulfate, inhibited the interactions. Glycosaminoglycan-free recombinant perlecan domain V and mutated domain I did not bind PRELP. The dissociation constants of the PRELP-perlecan interactions were in the range of 3-18 nm as determined by surface plasmon resonance. As expected, truncated PRELP, without the heparin-binding domain, did not bind perlecan. Confocal immunohistochemistry showed that PRELP outlines basement membranes with a location adjacent to perlecan. We also found that PRELP binds collagen type I and type II through its leucine-rich repeat domain. Electron microscopy visualized a complex with PRELP binding simultaneously to the triple helical region of procollagen I and the heparan sulfate chains of perlecan. Based on the location of PRELP and its interaction with perlecan heparan sulfate chains and collagen, we propose a function of PRELP as a molecule anchoring basement membranes to the underlying connective tissue.     

A heparin-binding growth factor, midkine, binds to a chondroitin sulfate proteoglycan, PG-M/versican.

Midkine is a heparin-binding growth factor with survival-promoting and migration-enhancing activities. In order to understand the regulation of midkine signaling, we isolated midkine-binding proteoglycans from day 13 mouse embryos, when midkine is intensely expressed. Deglycosylation followed by SDS/PAGE revealed various protein bands; one of these was identified as PG-M/versican by in gel trypsin digestion and sequencing the resulting peptides. PG-M/versican isolated from day 13 mouse embryos bound midkine with a Kd of 1.0 nM. Pleiotrophin/heparin-binding growth-associated molecule, which has a structure related to midkine, was also bound similarly. Digestion with chondroitinase ABC, AC-I or B abolished the binding to midkine. Heparin as well as chondroitin sulfate D and E inhibited the binding. After chondroitinase ABC digestion, the midkine-binding PG-M/versican released 4-sulfated, 6-sulfated, 2, 6-disulfated and 4,6-disulfated unsaturated disaccharides. These results suggest that midkine binds to a polysulfated domain in the chondroitin sulfate chain with a region of dermatan sulfate structure. This proteoglycan may modulate the midkine activity, as binding to midkine can enhance midkine action by concentrating it to the cell periphery or inhibit the action by competing with the binding to a signaling receptor.     

Coating electrospun collagen and gelatin fibers with perlecan domain I for increased growth factor binding

Electrospun natural polymer membranes were fabricated from collagen or gelatin coated with a bioactive recombinant fragment of perlecan, a natural heparan sulfate proteoglycan. The electrospinning process allowed the facile processing of a three-dimensional, porous fibril (2-6 microm in diameter) matrix suitable for tissue engineering. Laser scanning confocal microscopy revealed that osteoblast-like MG63 cells infiltrated the depth of the electrospun membrane evenly without visible apoptosis. Tissue engineering scaffolds ideally mimic the extracellular matrix; therefore, the electrospun membrane must contain both structural and functional matrix features. Fibers were coated, after processing, with perlecan domain I (PlnDI) to improve binding of basic fibroblast growth factor (FGF-2), which binds to native heparan sulfate chains on PlnDI. PlnDI-coated electrospun collagen fibers were ten times more effective than heparin-BSA collagen fibers at binding FGF-2. Because FGF-2 modulates cell growth, differentiation, migration and survival, the ability to effectively bind FGF-2 to an electrospun matrix is a key improvement in creating a successful tissue engineering scaffold.     

Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation

Perlecan (Hspg2) is a heparan sulfate proteoglycan expressed in basement membranes and cartilage. Perlecan deficiency (Hspg2(-/-)) in mice and humans causes lethal chondrodysplasia, which indicates that perlecan is essential for cartilage development. However, the function of perlecan in endochondral ossification is not clear. Here, we report the critical role of perlecan in VEGF signaling and angiogenesis in growth plate formation. The Hspg2(-/-) growth plate was significantly wider but shorter due to severely impaired endochondral bone formation. Hypertrophic chondrocytes were differentiated in Hspg2(-/-) growth plates; however, removal of the hypertrophic matrix and calcified cartilage was inhibited. Although the expression of MMP-13, CTGF, and VEGFA was significantly upregulated in Hspg2(-/-) growth plates, vascular invasion into the hypertrophic zone was impaired, which resulted in an almost complete lack of bone marrow and trabecular bone. We demonstrated that cartilage perlecan promoted activation of VEGF/VEGFR by binding to the VEGFR of endothelial cells. Expression of the perlecan transgene specific to the cartilage of Hspg2(-/-) mice rescued their perinatal lethality and growth plate abnormalities, and vascularization into the growth plate was restored, indicating that perlecan in the growth plate, not in endothelial cells, is critical in this process. These results suggest that perlecan in cartilage is required for activating VEGFR signaling of endothelial cells for vascular invasion and for osteoblast migration into the growth plate. Thus, perlecan in cartilage plays a critical role in endochondral bone formation by promoting angiogenesis essential for cartilage matrix remodeling and subsequent endochondral bone formation.     

Targeting perlecan in human keratinocytes reveals novel roles for perlecan in epidermal formation

Heparin-binding growth factors are crucial for the formation of human epidermis, but little is known about the role of heparan sulfate proteoglycans in this process. Here we investigated the role of the heparan sulfate proteoglycan, perlecan, in the formation of human epidermis, by utilizing in vitro engineered human skin. By disrupting perlecan expression either in the dermis or the epidermis, we found that epidermally derived perlecan is essential for epidermal formation. Perlecan-deficient keratinocytes formed a strikingly thin and poorly organized epidermis because of premature apoptosis and failure to complete their stratification program. Exogenous perlecan fully restored epidermal formation. Perlecan deposition in the basement membrane zone correlated with formation of multilayered epidermis. Perlecan deficiency, however, had no effect on the lining and deposition of major basement membrane components as was evident by a continuous linear staining of laminin and collagen IV. Similarly, perlecan deficiency did not affect the distribution of beta1 integrin. Addition of the perlecan ligand, fibroblast growth factor 7, protected perlecan-deficient keratinocytes from cell death and improved the thickness of the epidermis. Taken together, our results revealed novel roles for perlecan in epidermal formation. Perlecan regulates both the survival and terminal differentiation steps of keratinocytes. Our results suggested a model whereby perlecan regulates these processes via controlling the bioavailability of perlecan-binding soluble factors involved in epidermal morphogenesis.     

Characterization of a low-sulfated chondroitin sulfate from the body of Viviparus ater (mollusca gastropoda). Modification of its structure by lead pollution

A chondroitin sulfate was purified from the body of Viviparus ater (Mollusca gastropoda) and analyzed for molecular mass, constituent disaccharides, and structure by 1H NMR and 1H 2D NMR. A quite unique glycosaminoglycan species was isolated having a high molecular mass (greater than 45,000) and low charge density, about 0.60, due to the presence of 42% non-sulfated disaccharide, 5% 6-sulfated disaccharide, 48% 4-sulfated disaccharide, and 5% 4,6-disulfated disaccharide. Specimens of Mollusca were also submitted to lead exposure for different times, and the effect on chondroitin sulfate structure was studied. After 96 h treatment a strong decrease in chondroitin sulfate content was observed with a significant modification of its structure producing a more desulfated polymer, in particular in position 4 of the galactosamine unit. Simultaneously, the amount of unsaturated non-sulfated disaccharide increased with an overall decrease of the charge density.     

Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor.

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.     

The structure, location, and function of perlecan, a prominent pericellular proteoglycan of fetal, postnatal, and mature hyaline cartilages

The aim of this study was to immunolocalize perlecan in human fetal, postnatal, and mature hyaline cartilages and to determine information on the structure and function of chondrocyte perlecan. Perlecan is a prominent component of human fetal (12-14 week) finger, toe, knee, and elbow cartilages; it was localized diffusely in the interterritorial extracellular matrix, densely in the pericellular matrix around chondrocytes, and to small blood vessels in the joint capsules and perichondrium. Aggrecan had a more intense distribution in the marginal regions of the joint rudiments and in para-articular structures. Perlecan also had a strong pericellular localization pattern in postnatal (2-7 month) and mature (55-64 year) femoral cartilages, whereas aggrecan had a prominent extracellular matrix distribution in these tissues. Western blotting identified multiple perlecan core protein species in extracts of the postnatal and mature cartilages, some of which were substituted with heparan sulfate and/or chondroitin sulfate and some were devoid of glycosaminoglycan substitution. Some perlecan core proteins were smaller than intact perlecan, suggesting that proteolytic processing or alternative splicing had occurred. Surface plasmon resonance and quartz crystal microbalance with dissipation experiments demonstrated that chondrocyte perlecan bound fibroblast growth factor (FGF)-1 and -9 less efficiently than endothelial cell perlecan. The latter perlecan supported the proliferation of Baf-32 cells transfected with FGFR3c equally well with FGF-1 and -9, whereas chondrocyte perlecan only supported Baf-32 cell proliferation with FGF-9. The function of perlecan therefore may not be universal but may vary with its cellular origin and presumably its structure.     

Amyloid-beta interactions with chondroitin sulfate-derived monosaccharides and disaccharides. implications for drug development

In Alzheimer's disease, the major pathological features are diffuse and senile plaques that are primarily composed of the amyloid-beta (A beta) peptide. It has been proposed that proteoglycans and glycosaminoglycans (GAG) facilitate amyloid fibril formation and/or stabilize the plaque aggregates. To develop effective therapeutics based on A beta-GAG interactions, understanding the A beta binding motif on the GAG chain is imperative. Using electron microscopy, fluorescence spectroscopy, and competitive inhibition ELISAs, we have evaluated the ability of chondroitin sulfate-derived monosaccharides and disaccharides to induce the structural changes in A beta that are associated with GAG interactions. Our results demonstrate that the disaccharides GalNAc-4-sulfate(4S), Delta UA-GalNAc-6-sulfate(6S), and Delta UA-GalNAc-4,6-sulfate(4S,6S), the iduronic acid-2-sulfate analogues, and the monosaccharides d-GalNAc-4S, d-GalNAc-6S, and d-GalNAc-4S,6S, but not d-GalNAc, d-GlcNAc, or Delta UA-GalNAc, induce the fibrillar features of A beta-GAG interactions. The binding affinities of all chondroitin sulfate-derived saccharides mimic those of the intact GAG chains. The sulfated monosaccharides and disaccharides compete with the intact chondroitin sulfate and heparin GAGs for A beta binding, as illustrated by competitive inhibition ELISAs. Therefore, the development of therapeutics based on the model of A beta-chondroitin sulfate binding may lead to effective inhibitors of the GAG-induced amyloid formation that is observed in vitro.     

A Monoclonal Antibody which Recognizes a Glycosaminoglycan Epitope in Both Dermatan Sulfate and Chondroitin Sulfate Proteoglycans of Human Skin

Studies have been initiated to identify various cell surface and matrix components of normal human skin through the production and characterization of murine monoclonal antibodies. One such antibody, termed PG-4, identifies both cell surface and matrix antigens in extracts of human foetal and adult skin as the dermatan sulfate proteoglycans, decorin and biglycan, and the chondroitin sulfate proteoglycan versican. Treatment of proteoglycans with chondroitinases completely abolishes immunoreactivity for all of these antigens which suggests that the epitope resides within their glycosaminoglycan chains. Further evidence for the carbohydrate nature of the epitope derives from competition studies where protein-free chondroitin sulfate chains from shark cartilage react strongly; however, chondroitin sulfate chains from bovine tracheal cartilage fail to exhibit a significant reactivity, an indication that the epitope, although present in some chondroitin sulfate chains, does not consist of random chondroitin 4- or 6-sulfate disaccharides. The presence of the epitope on dermatan sulfate chains and on decorin was also demonstrated using competition assays. Thus, PG-4 belongs to a class of antibodies that recognize native epitopes located within glycosaminoglycan chains. It differs from previously described antibodies in this class in that it identifies both chondroitin sulfate and dermatan sulfate proteoglycans. These characteristics make PG-4 a useful monoclonal antibody probe to identify the total population of proteoglycans in human skin.     

Atherosclerosis in perlecan heterozygous mice

The hypothesis that lipoprotein association with perlecan is atherogenic was tested by studying atherosclerosis in mice that had a heterozygous deletion of perlecan, the primary extracellular heparan sulfate proteoglycan in arteries. We first studied the expression of perlecan in mouse lesions and noted that this proteoglycan in aorta was found in the subendothelial matrix. Perlecan was also a major component of the lesional extracellular matrix. Mice with a heterozygous deletion had a reduction in arterial wall perlecan expression. Atherosclerosis in these mice was studied after crossing the defect into the apolipoprotein E (apoE) and LDL receptor knockout backgrounds. At 12 weeks, chow-fed apoE null mice with a heterozygous deletion had less atherosclerosis. However, at 24 weeks and in the LDL receptor heterozygous background, the presence of a perlecan knockout allele did not significantly alter lesion size. Thus, it appears that loss of perlecan leads to less atherosclerosis in early lesions. Although this might be attributable to a decrease in lipoprotein retention, it should be noted that perlecan might mediate multiple other processes that could, in sum, accelerate atherosclerosis.     

The core protein of growth plate perlecan binds FGF-18 and alters its mitogenic effect on chondrocytes

Fibroblast growth factor-18 (FGF-18) has been shown to regulate the growth plate chondrocyte proliferation, hypertrophy and cartilage vascularization necessary for endochondral ossification. The heparan sulfate proteoglycan perlecan is also critical for growth plate chondrocyte proliferation. FGF-18 null mice exhibit a skeletal dwarfism similar to that of perlecan null mice. Growth plate perlecan contains chondroitin sulfate (CS) and heparan sulfate (HS) chains and FGF-18 is known to bind to heparin and to heparan sulfate from some sources. We used cationic filtration and immunoprecipitation assays to investigate the binding of FGF-18 to perlecan purified from the growth plate and to recombinant perlecan domains expressed in COS-7 cells. FGF-18 bound to perlecan with a K_d of 145 nM. Near saturation, ∼103 molecules of FGF-18 bound per molecule of perlecan. At the lower concentrations used, FGF-18 bound with a K_d of 27.8 nM. This binding was not significantly altered by chondroitinase nor heparitinase digestion of perlecan, but was substantially and significantly reduced by reduction and alkylation of the perlecan core protein. This indicates that the perlecan core protein (and not the CS nor HS chains) is involved in FGF-18 binding. FGF-18 bound equally to full-length perlecan purified from the growth plate and to recombinant domains I-III and III of perlecan. These data indicate that low affinity binding sites for FGF-18 are present in cysteine-rich regions of domain III of perlecan. FGF-18 stimulated ³H-thymidine incorporation in growth plate chondrocyte cultures derived from the lower and upper proliferating zones by 9- and 14-fold, respectively. The addition of perlecan reversed this increased incorporation in the lower proliferating chondrocytes by 74% and in the upper proliferating cells by 37%. These results suggest that perlecan can bind FGF-18 and alter the mitogenic effect of FGF-18 on growth plate chondrocytes.     

Expression of the heparan sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro.

Expression of the basement membrane heparan sulfate proteoglycan (HSPG), perlecan (Pln), mRNA, and protein has been examined during murine development. Both Pln mRNA and protein are highly expressed in cartilaginous regions of developing mouse embryos, but not in areas of membranous bone formation. Initially detected at low levels in precartilaginous areas of d 12.5 embryos, Pln protein accumulates in these regions through d 15.5 at which time high levels are detected in the cartilage primordia. Laminin and collagen type IV, other basal lamina proteins commonly found colocalized with Pln, are absent from the cartilage primordia. Accumulation of Pln mRNA, detected by in situ hybridization, was increased in d 14.5 embryos. Cartilage primordia expression decreased to levels similar to that of the surrounding tissue at d 15.5. Pln accumulation in developing cartilage is preceded by that of collagen type II. To gain insight into Pln function in chondrogenesis, an assay was developed to assess the potential inductive activity of Pln using multipotential 10T1/2 murine embryonic fibroblast cells. Culture on Pln, but not on a variety of other matrices, stimulated extensive formation of dense nodules reminiscent of embryonic cartilaginous condensations. These nodules stained intensely with Alcian blue and collagen type II antibodies. mRNA encoding chondrocyte markers including collagen type II, aggrecan, and Pln was elevated in 10T1/2 cells cultured on Pln. Human chondrocytes that otherwise rapidly dedifferentiate during in vitro culture also formed nodules and expressed high levels of chondrocytic marker proteins when cultured on Pln. Collectively, these studies demonstrate that Pln is not only a marker of chondrogenesis, but also strongly potentiates chondrogenic differentiation in vitro.     

Glycosaminoglycan structures required for strong binding to midkine,a heparin-binding growth factor

Midkine (MK), a heparin-binding growth factor, binds strongly to oversulfated structures in chondroitin sulfates (CSs) and heparan sulfate. To elucidate the carbohydrate structure actually involved in the strong binding, dissected brains from 13-day mouse embryos were incubated with [14C]-glucosamine. The labeled glycosaminoglycans were fractionated by MK-agarose affinity chromatography to a weakly binding fraction, which was eluted by 0.5 M NaCl, and a strongly binding fraction, which was eluted by higher NaCl concentrations. Among the unsaturated disaccharides released from the strongly binding fraction by chondroitinase ABC, DeltaDi-diSE with 4,6-disulfated N-acetylgalactosamine accounted for 32.3%, whereas its content was lower in the weakly binding fraction. Artificial CS-E structure was formed using N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid or recombinant human enzyme. Analysis of the products and their interaction with MK revealed that E units without 3-O-sulfation of glucuronic acid are sufficient for strong binding, provided that they are present as a dense cluster. Among the sulfated disaccharides released by heparitinase digestion, the trisulfated one, DeltaDiHS-triS, was the most abundant in the strongly binding fraction and was lower in the weakly binding fraction. Together with results of previous studies, we concluded that the multivalent trisulfated heparin-like unit is another structure involved in strong binding to MK.     

Midkine and pleiotrophin: two related proteins involved in development,survival, inflammation and tumorigenesis

Midkine (MK) and pleiotrophin (PTN) are low molecular weight proteins with closely related structures. They are mainly composed of two domains held by disulfide bridges, and there are three antiparallel beta-sheets in each domain. MK and PTN promote the growth, survival, and migration of various cells, and play roles in neurogenesis and epithelial mesenchymal interactions during organogenesis. A chondroitin sulfate proteoglycan, protein-tyrosine phosphatase zeta (PTPzeta), is a receptor for MK and PTN. The downstream signaling system includes ERK and PI3 kinase. MK binds to the chondroitin sulfate portion of PTPzeta with high affinity. Among the various chondroitin sulfate structures, the E unit, which has 4,6-disulfated N-acetylgalactosamine, provides the strongest binding site. The expression of MK and PTN is increased in various human tumors, making them promising as tumor markers and as targets for tumor therapy. MK and PTN expression also increases upon ischemic injury. MK enhances the migration of inflammatory cells, and is involved in neointima formation and renal injury following ischemia. MK is also interesting from the viewpoints of the treatment of neurodegenerative diseases, increasing the efficiency of in vitro development, and the prevention of HIV infection.     

Localization of glycosaminoglycan substitution sites on domain V of mouse perlecan

Perlecan, the predominant basement membrane proteoglycan, has previously been shown to contain glycosaminoglycans attached at serine residues, numbers 65, 71, and 76, in domain I. However, the C-terminal domains IV and V of this molecule may also be substituted with glycosaminoglycan chains, but the exact substitution sites were not identified. The amino acid sequence of mouse perlecan reveals many ser-gly sequences in these domains that are possible sites for glycosaminoglycan substitution. We expressed recombinant domain IV and/or V of mouse perlecan in COS-7 cells and analyzed glycosaminoglycan substitution. Both heparan sulfate and chondroitin sulfate chains could be detected on recombinant domain V. One site, ser-gly-glu (serine residue 3593), toward the C-terminal region of domain V is a substitution site for heparan sulfate. When this sequence was absent, chondroitin/dermatan sulfate substitution was deleted, and the likely site for this galactosaminoglycan substitution was ser-gly-ala-gly (serine residue 3250) on domain V.