Structural basis of glycosaminoglycan modification and of heterotypic interactions of perlecan domain V.

The C-terminal perlecan domain V of about 90 kDa consists of laminin-type G domain modules (LG) (25 kDa) and epidermal growth factor-like modules (EG) (4 kDa) in the tandem arrangement LG1-EG1-EG2-LG2-EG3-EG4-LG3. Several shorter fragments have been prepared by recombinant production in mammalian cells and used to map the single glycosaminoglycan (GAG) substitution site and the binding of several carbohydrate and protein ligands. This identified a Ser3511 residue located in a short link region between EG4 and LG3 as being involved in GAG attachment. Electron microscopy provided evidence that the same substitution exists in tissue forms of perlecan. Heparan sulphate attached to this site was shown to bind to the alpha1LG4 module of laminin-1, indicating a role in basement membrane assembly and cell-matrix interactions. This site is also close to an Asn-Asp bond which is readily cleaved by an endogenous protease that depends on the presence of Asp and the LG2 module. A weak heparin binding site was shown to include the EG2 module, which contains five basic residues. Binding to sulphatides and the alpha-dystroglycan receptor was much stronger and required at least two LG modules. However, single LG modules appear to be sufficient for the interaction with the laminin-nidogen complex, while EG3-4 and some flanking regions are apparently involved in fibulin-2 binding. These observations indicate that a complex modular structure is required for domain V in order to provide a rich repertoire of potential biological functions.     

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.     

BMP-1/Tolloid-like metalloproteases process endorepellin, the angiostatic C-terminal fragment of perlecan

Endorepellin, the C-terminal domain of the heparan sulfate proteoglycan perlecan, possesses angiostatic activity. The terminal laminin-like globular (LG3) domain of endorepellin appears to possess most of the biological activity on endothelial cells. LG3 protein has been detected in the urine of patients with end-stage renal disease and in the amniotic fluid of pregnant women with premature rupture of fetal membranes. These findings suggest that proteolytic processing of endorepellin and the generation of LG3 might have biological significance. In this study, we have identified specific enzymes of the bone morphogenetic protein-1 (BMP-1)/Tolloid family of metalloproteases that cleave LG3 from recombinant endorepellin at the physiologically relevant site and that cleave LG3 from endogenous perlecan in cultured mouse and human cells. The BMP-1/Tolloid family of metalloproteases is thereby implicated in the processing of a major basement membrane proteoglycan and in the liberation of an anti-angiogenic factor. Using molecular modeling, site-directed mutagenesis and angiogenic assays, we further demonstrate that LG3 activity requires specific amino acids involved in Ca(2+) coordination.     

Perlecan protein core interacts with extracellular matrix protein 1 (ECM1), a glycoprotein involved in bone formation and angiogenesis

The goal of this study was to discover novel partners for perlecan, a major heparan sulfate proteoglycan of basement membranes, and to examine new interactions through which perlecan may influence cell behavior. We employed the yeast two-hybrid system and used perlecan domain V as bait to screen a human keratinocyte cDNA library. Among the strongest interacting clones, we isolated a approximately 1.6-kb cDNA insert that encoded extracellular matrix protein 1 (ECM1), a secreted glycoprotein involved in bone formation and angiogenesis. The sequencing of the clone revealed the existence of a novel splice variant that we name ECM1c. The interaction was validated by co-immunoprecipitation studies, using both cell-free systems and mammalian cells, and the specific binding site within each molecule was identified employing various deletion mutants. The C terminus of ECM1 interacted specifically with the epidermal growth factor-like modules flanking the LG2 subdomain of perlecan domain V. Perlecan and ECM1 were also co-expressed by a variety of normal and transformed cells, and immunohistochemical studies showed a partial expression overlap, particularly around dermal blood vessels and adnexal epithelia. ECM1 has been shown to regulate endochondral bone formation, stimulate the proliferation of endothelial cells, and induce angiogenesis. Similarly, perlecan plays an important role in chondrogenesis and skeletal development, as well as harboring pro- and anti-angiogenic activities. Thus, a physiological interaction could also occur in vivo during development and in pathological events, including tissue remodeling and tumor progression.     

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.     

Perlecan and tumor angiogenesis.

Perlecan is a major heparan sulfate proteoglycan (HSPG) of basement membranes (BMs) and connective tissues. The core protein of perlecan is divided into five domains based on sequence homology to other known proteins. Commonly, the N-terminal domain I of mammalian perlecan is substituted with three HS chains that can bind a number of matrix molecules, cytokines, and growth factors. Perlecan is essential for metazoan life, as shown by genetic manipulations of nematodes, insects, and mice. There are also known human mutations that can be lethal. In vertebrates, new functions of perlecan emerged with the acquisition of a closed vascular system and skeletal connective tissues. Many of perlecan's functions may be related to the binding and presentation of growth factors to high-affinity tyrosine kinase (TK) receptors. Data are accumulating, as discussed here, that similar growth factor-mediated processes may have unwanted promoting effects on tumor cell proliferation and tumor angiogenesis. Understanding of these attributes at the molecular level may offer opportunities for therapeutic intervention.     

Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins

The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly.     

Essential contribution of tumor-derived perlecan to epidermal tumor growth and angiogenesis.

As a major heparan sulfate proteoglycan (PG) in basement membranes, perlecan has been linked to tumor invasion, metastasis, and angiogenesis. Here we produced epidermal tumors in immunocompromised rats by injection of mouse RT101 tumor cells. Tumor sections stained with species-specific perlecan antibodies, together with immunoelectron microscopy, showed that perlecan distributed around blood vessels was of both host and tumor cell origin. Tumor-derived perlecan was also distributed throughout the tumor matrix. Blood vessels stained with rat-specific PECAM-1 antibody showed their host origin. RT101 cells also expressed two other basement membrane heparan sulfate PGs, agrin and type XVIII collagen. Antisense targeting of perlecan inhibited tumor cell growth in vitro, while exogenous recombinant perlecan, but not heparin, restored the growth of antisense perlecan-expressing cells, suggesting that perlecan core protein, rather than heparan sulfate chains from perlecan, agrin, or type XVIII collagen, regulates tumor cell growth. However, perlecan core protein requirement was not related to fibroblast growth factor-7 binding because RT101 cells were unresponsive to and lacked receptors for this growth factor. In vivo, antisense perlecan-transfected cells generated no tumors, whereas untransfected and vector-transfected cells formed tumors with obvious neovascularization, suggesting that tumor perlecan rather than host perlecan controls tumor growth and angiogenesis.     

Extracellular matrices of the avian ovarian follicle. Molecular characterization of chicken perlecan

In egg-laying species, such as the chicken, the mode of transport of lipoprotein particles from the capillary plasma to endocytic receptors on the oocyte surface is largely unknown. Here we show by molecular characterization that the large prominent heparan sulfate proteoglycan of extracellular matrices, termed perlecan or HSPG2 (the product of the hspg2 gene), is a component of ovarian follicles that may participate in this process. However, although normally a major HSPG of basement membranes or basal laminae, in chicken follicles, perlecan is absent from the membranous structure between the theca interna and granulosa cell layers, which to date has been considered a bona fide basement membrane. Rather, the protein is localized in the extracellular matrix of theca externa cells, which produce this HSPG. Furthermore, in chicken testes, perlecan is localized in the peritubular spaces but in less organized fashion than the classical basement membrane components, agrin and laminin. All five domains and structural hallmarks of chicken perlecan (4071 residues) have been conserved in its mammalian counterparts. We have produced the recombinant domain II (containing low density lipoprotein (LDL) receptor-like binding repeats) of chicken perlecan and demonstrate its capacity to bind LDL and very low density lipoprotein (VLDL), apolipoprotein B-containing lipoproteins ultimately destined for uptake into oocytes via members of the low density lipoprotein receptor family. Binding to perlecan heparan sulfate side chains may facilitate the interaction of lipoproteins with domain II. Based on the current results and on domain-domain interactions revealed by recent ultrastructural investigations of the LDL receptor, nidogen, and laminin (Rudenko, G., Henry, L., Henderson, K., Ichtchenko, K., Brown, M. S., Goldstein, J. L., and Deisenhofer, J. (2002) Science 298, 2353-2358 and Takagi, J., Yang, Y., Liu, J. H., Wang, J. H., and Springer, T. A. (2003) Nature 424, 969-974), we propose a novel role of perlecan in mediating plasma-to-oocyte surface transport of VLDL particles.     

Perlecan and its immunoglobulin like domain IV are abundant in vitreous and serum of the chick embryo.

Perlecan is a highly conserved heparan sulfate proteoglycan in cartilage and basement membranes. We identified chick perlecan and a 90 KD perlecan fragment in vivo using a newly generated monoclonal antibody. Chick perlecan is, like its human and mouse homologue, a hybrid heparan sulfate/chondroitin sulfate proteoglycan with a core protein of 400 KD. Analysis of the 90 KD fragment by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and Capillary LC nano Electrospray Ionization tandem MS (LC nano ESI MS/MS) showed that it belonged to domain IV of the perlecan core protein. We found that full-length perlecan and its domain IV fragment are abundant in embryonic vitreous body and serum. Their expression in vitreous and serum is greatly down-regulated shortly after hatching of the chick. We speculate that the abundance of perlecan in the embryonic circulation and vitreous reflects the ongoing formation of new BMs in the expanding vascular system and the growing retina. In addition, we found that perlecan as a substrate does not support, rather inhibits neurite outgrowth.     

Localization of perlecan and heparanase in Hertwig's epithelial root sheath during root formation in mouse molars.

During cementogenesis, dental follicular cells penetrate the ruptured Hertwig's epithelial root sheath (HERS) and differentiate into cementoblasts. Mechanisms involved in basement membrane degradation during this process have not been clarified. Perlecan, a heparan sulfate (HS) proteoglycan, is a component of all basement membranes. Degradation of HS of perlecan by heparanase cleavage affects a variety of biological processes. We elucidated immunolocalization of perlecan and heparanase in developing murine molars to clarify their roles in cementoblast differentiation. At the initial stage of root formation, perlecan immunoreactivity was detected on the basement membrane of HERS. Weak heparanase immunoreactivity was detected in HERS cells. HERS showed intense staining for heparanase as root formation progressed. In contrast, labeling for perlecan disappeared from the basement membrane facing the dental follicle, and weak immunoreactivity for perlecan was detected on the inner side of the basement membrane of HERS. These findings suggest that perlecan removal is an important step for root and periodontal tissue formation. Heparanase secreted by the cells of HERS may contribute to root formation by degrading perlecan in the dental basement membrane.     

Endothelial cells interact with the core protein of basement membrane perlecan through beta 1 and beta 3 integrins: an adhesion modulated by glycosaminoglycan

Aortic endothelial cells adhere to the core protein of murine perlecan, a heparan sulfate proteoglycan present in endothelial basement membrane. We found that cell adhesion was partially inhibited by beta 1 integrin-specific mAb and almost completely blocked by a mixture of beta 1 and alpha v beta 3 antibodies. Furthermore, adhesion was partially inhibited by a synthetic peptide containing the perlecan domain III sequence LPASFRGDKVTSY (c-RGD) as well as by GRGDSP, but not by GRGESP. Both antibodies contributed to the inhibition of cell adhesion to immobilized c-RGD whereas only beta 1-specific antibody blocked residual cell adhesion to proteoglycan core in the presence of maximally inhibiting concentrations of soluble RGD peptide. A fraction of endothelial surface-labeled detergent lysate bound to a core affinity column and 147-, 116-, and 85-kD proteins were eluted with NaCl and EDTA. Polyclonal anti-beta 1 and anti-beta 3 integrin antibodies immunoprecipitated 116/147 and 85/147 kD surface-labeled complexes, respectively. Cell adhesion to perlecan was low compared to perlecan core, and cell adhesion to core, but not to immobilized c-RGD, was selectively inhibited by soluble heparin and heparan sulfates. This inhibition by heparin was also observed with laminin and fibronectin and, in the case of perlecan, was found to be independent of heparin binding to substrate. These data support the hypothesis that endothelial cells interact with the core protein of perlecan through beta 1 and beta 3 integrins, that this binding is partially RGD- independent, and that this interaction is selectively sensitive to a cell-mediated effect of heparin/heparan sulfates which may act as regulatory ligands.     

Human basement membrane heparan sulfate proteoglycan core protein: a 467-kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor

The primary structure of the large human basement membrane heparan sulfate proteoglycan (HSPG) core protein was determined from cDNA clones. The cDNA sequence codes for a 467-kD protein with a 21-residue signal peptide. Analysis of the amino acid sequence showed that the protein consists of five domains. The amino-terminal domain I contains three putative heparan sulfate attachment sites; domain II has four LDL receptor-like repeats; domain III contains repeats similar to those in the short arms of laminin; domain IV has lg-like repeats resembling those in neural cell adhesion molecules; and domain V contains sequences resembling repeats in the G domain of the laminin A chain and repeats in the EGF. The domain structure of the human basement membrane HSPG core protein suggests that this mosaic protein has evolved through shuffling of at least four different functional elements previously identified in other proteins and through duplication of these elements to form the functional domains. Comparison of the human amino acid sequence with a partial amino acid sequence from the corresponding mouse protein (Noonan, D. M., E. A. Horigan, S. R. Ledbetter, G. Vogeli, M. Sasaki, Y. Yamada, and J. R. Hassell. 1988. J. Biol. Chem. 263:16379-16387) shows a major difference between the species in domain IV, which contains the Ig repeats: seven additional repeats are found in the human protein inserted in the middle of the second repeat in the mouse sequence. This suggests either alternative splicing or a very recent duplication event in evolution. The multidomain structure of the basement membrane HSPG implies a versatile role for this protein. The heparan sulfate chains presumably participate in the selective permeability of basement membranes and, additionally, the core protein may be involved in a number of biological functions such as cell binding, LDL-metabolism, basement membrane assembly, calcium binding, and growth- and neurite-promoting activities.     

Matrilysin/matrix metalloproteinase-7(MMP7) cleavage of perlecan/HSPG2 creates a molecular switch to alter prostate cancer cell behavior

Perlecan/HSPG2, a large heparan sulfate (HS) proteoglycan, normally is expressed in the basement membrane (BM) underlying epithelial and endothelial cells. During prostate cancer (PCa) cell invasion, a variety of proteolytic enzymes are expressed that digest BM components including perlecan. An enzyme upregulated in invasive PCa cells, matrilysin/matrix metalloproteinase-7 (MMP-7), was examined as a candidate for perlecan proteolysis both in silico and in vitro. Purified perlecan showed high sensitivity to MMP-7 digestion even when fully decorated with HS or when presented in native context connected with other BM proteins. In both conditions, MMP-7 produced discrete perlecan fragments corresponding to an origin in immunoglobulin (Ig) repeat region domain IV. While not predicted by in silico analysis, MMP-7 cleaved every subpart of recombinantly generated perlecan domain IV. Other enzymes relevant to PCa that were tested had limited ability to cleave perlecan including prostate specific antigen, hepsin, or fibroblast activation protein α. A long C-terminal portion of perlecan domain IV, Dm IV-3, induced a strong clustering phenotype in the metastatic PCa cell lines, PC-3 and C4-2. MMP-7 digestion of Dm IV-3 reverses the clustering effect into one favoring cell dispersion. In a C4-2 Transwell® invasion assay, perlecan-rich human BM extract that was pre-digested with MMP-7 showed loss of barrier function and permitted a greater level of cell penetration than untreated BM extract. We conclude that enzymatic processing of perlecan in the BM or territorial matrix by MMP-7 as occurs in the invasive tumor microenvironment acts as a molecular switch to alter PCa cell behavior and favor cell dispersion and invasiveness.     

The extracellular matrix protein WARP is a novel component of a distinct subset of basement membranes.

WARP is a recently described member of the von Willebrand factor A domain superfamily of extracellular matrix proteins, and is encoded by the Vwa1 gene. We have previously shown that WARP is a multimeric component of the chondrocyte pericellular matrix in articular cartilage and intervertebral disc, where it interacts with the basement membrane heparan sulfate proteoglycan perlecan. However, the tissue-specific expression of WARP in non-cartilaginous tissues and its localization in the extracellular matrix of other perlecan-containing tissues have not been analyzed in detail. To visualize WARP-expressing cells, we generated a reporter gene knock-in mouse by targeted replacement of the Vwa1 gene with beta-galactosidase. Analysis of reporter gene expression and WARP protein localization by immunostaining demonstrates that WARP is a component of a limited number of distinct basement membranes. WARP is expressed in the vasculature of neural tissues and in basement membrane structures of the peripheral nervous system. Furthermore, WARP is also expressed in the apical ectodermal ridge of developing limb buds, and in skeletal and cardiac muscle. These findings are the first evidence for WARP expression in non-cartilaginous tissues, and the identification of WARP as a component of a limited range of specialized basement membranes provides further evidence for the heterogeneous composition of basement membranes between different tissues.     

Crystal structure and mutational analysis of a perlecan-binding fragment of nidogen-1.

Nidogen, an invariant component of basement membranes, is a multifunctional protein that interacts with most other major basement membrane proteins. Here, we report the crystal structure of the mouse nidogen-1 G2 fragment, which contains binding sites for collagen IV and perlecan. The structure is composed of an EGF-like domain and an 11-stranded beta-barrel with a central helix. The beta-barrel domain has unexpected similarity to green fluorescent protein. A large surface patch on the beta-barrel is strikingly conserved in all metazoan nidogens. Site-directed mutagenesis demonstrates that the conserved residues are involved in perlecan binding.     

Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin.

The C-terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non-covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25-32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N-terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin-1 and fibulin-2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin-1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22-38 kDa) and in serum (120-300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N-terminal association region (approximately 50 residues), a central protease-sensitive hinge region (approximately 70 residues) and a C-terminal stable endostatin domain (approximately 180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix-associated to a more soluble endocrine form.