Molecular characteristics of fibroblast growth factor–fibroblast growth factor receptor–heparin-like glycosaminoglycan complex

Fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. Understanding of the molecular nature of interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF–FGFR complex. In this study, based on an exhaustive analysis of the primary sequences of the FGF family, we determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. We propose that the FGF–FGFR interaction mediated by the ‘conserved’ primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the ‘variable’ secondary sites, on both FGF as well as FGFR mediates specificity of a given FGF to a given FGFR isoform. Furthermore, as the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, we find that the spatial orientation of the primary receptor-binding site of FGF-2 coincides structurally with the IL-1β receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF–FGFR interactions. In the FGF–FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG.

Enhanced Glycosylation and Sulfation of Secretory Proteoglycans Is Coupled to the Expression of a Basic Secretory Protein

We have used coexpression of a salivary basic proline-rich protein (PRP) along with a proline-rich proteoglycan (PRPg) in pituitary AtT-20 cells to examine the regulation of glycosaminoglycan (GAG) biosynthesis and the storage of these secretory products for regulated secretion. The basic PRP caused a dose-dependent increase in sulfation of PRPg and also increased the extent to which PRPg polypeptide backbones are modified by a GAG chain. The sulfation of an endogenous proteoglycan was similarly increased in the presence of basic PRP; however, other sulfated secretory products of AtT-20 cells were unaffected. These results imply that enzymes functioning in elongation and sulfation of proteoglycans are coordinately regulated and that their activities respond to a change in the milieu of the intracellular transport pathway. Analysis of the regulated secretion of both the basic PRP and PRPg has indicated that while the presence of the GAG chain improves the storage of PRPg, the presence of PRPg does not increase the storage of basic PRP. Therefore, sulfation of GAGs does not appear to be a primary factor in regulated secretory sorting.

Cytoskeletal Reorganization Induced by Engagement of the NG2 Proteoglycan Leads to Cell Spreading and Migration

Cells expressing the NG2 proteoglycan can attach, spread, and migrate on surfaces coated with NG2 mAbs, demonstrating that engagement of NG2 can trigger the cytoskeletal rearrangements necessary for changes in cell morphology and motility. Engagement of different epitopes of the proteoglycan results in distinct forms of actin reorganization. On mAb D120, the cells contain radial actin spikes characteristic of filopodial extension, whereas on mAb N143, the cells contain cortical actin bundles characteristic of lamellipodia. Cells that express NG2 variants lacking the transmembrane and cytoplasmic domains are unable to spread or migrate on NG2 mAb-coated surfaces, indicating that these portions of the molecule are essential for NG2-mediated signal transduction. Cells expressing an NG2 variant lacking the C-terminal half of the cytoplasmic domain can still spread normally on mAbs D120 and N143, suggesting that the membrane-proximal cytoplasmic segment is responsible for this process. In contrast, this variant migrates poorly on mAb D120 and exhibits abnormal arrays of radial actin filaments decorated with fascin during spreading on this mAb. The C-terminal portion of the NG2 cytoplasmic domain, therefore, may be involved in regulating molecular events that are crucial for cell motility.

sqv-3, -7, and -8, a set of genes affecting morphogenesis in Caenorhabditis elegans, encode enzymes required for glycosaminoglycan biosynthesis

sqv (squashed vulva) genes comprise a set of eight independent loci in Caenorhabditis elegans required zygotically for the invagination of vulval epithelial cells and maternally for normal oocyte formation and embryogenesis. Sequencing of sqv-3, sqv-7, and sqv-8 suggested a role for the encoded proteins in glycolipid or glycoprotein biosynthesis. Using a combination of in vitro analysis of SQV enzymatic activities, sqv+-mediated rescue of vertebrate cell lines, and biochemical characterization of sqv mutants, we show that sqv-3, -7, and -8 all affect the biosynthesis of glycosaminoglycans and therefore compromise the function of one specific class of glycoconjugates, proteoglycans. These findings establish the importance of proteoglycans and their associated glycosaminoglycans in epithelial morphogenesis and patterning during C. elegans development.

Molecular cloning of a human melanoma-associated chondroitin sulfate proteoglycan.

A human melanoma-associated chondroitin sulfate proteoglycan (MCSP), recognized by mAb 9.2.27, plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. We report here the molecular cloning and nucleotide sequencing of cDNA encoding the entire core protein of human MCSP and provide its deduced amino acid sequence. This core protein contains an open reading frame of 2322 aa, encompassing a large extracellular domain, a hydrophobic transmembrane region, and a relatively short cytoplasmic tail. Northern blot analysis indicated that MCSP cDNA probes detect a single 8.0-kb RNA species expressed in human melanoma cell lines. In situ hybridization experiments with a segment of the MCSP coding sequence localized MCSP mRNA in biopsies prepared from melanoma skin metastases. Multiple human Northern blots with an MCSP-specific probe revealed a strong hybridization signal only with melanoma cells and not with other human cancer cells or a variety of human fetal and adult tissues. These data indicate that MCSP represents an integral membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells. The availability of cDNAs encoding MCSP should facilitate studies designed to establish correlations between structure and function of this molecule and help to establish its role in the progression of human malignant melanoma.

Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme.

Certain matrix metalloproteinases (MMP) are expressed within the fibrous areas surrounding acellular lipid cores of atherosclerotic plaques, suggesting that these proteinases degrade matrix proteins within these areas and weaken the structural integrity of the lesion. We report that matrilysin and macrophage metalloelastase, two broad-acting MMPs, were expressed in human atherosclerotic lesions in carotid endarterectomy samples (n = 18) but were not expressed in normal arteries (n = 7). In situ hybridization and immunohistochemistry revealed prominent expression of matrilysin in cells confined to the border between acellular lipid cores and overlying fibrous areas, a distribution distinct from other MMPs found in similar lesions. Metalloelastase was expressed in these same border areas. Matrilysin was present in lipid-laden macrophages, identified by staining with anti-CD-68 antibody. Furthermore, endarterectomy tissue in organ culture released matrilysin. Staining for versican demonstrated that this vascular proteoglycan was present at sites of matrilysin expression. Biochemical studies showed that matrilysin degraded versican much more efficiently than other MMPs present in atherosclerotic lesions. Our findings suggest that matrilysin, specifically expressed in atherosclerotic lesions, could cleave structural proteoglycans and other matrix components, potentially leading to separation of caps and shoulders from lipid cores.

Defining the domains of type I collagen involved in heparin- binding and endothelial tube formation

Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiquitous cell adhesion mechanism. However, the HSPG binding sites on type I collagen are unknown. Previously we mapped heparin binding to the vicinity of the type I collagen N terminus by electron microscopy. The present study has identified type I collagen sequences used for heparin binding and endothelial cell–collagen interactions. Using affinity coelectrophoresis, we found heparin to bind as follows: to type I collagen with high affinity (Kd ≈ 150 nM); triple-helical peptides (THPs) including the basic N-terminal sequence α1(I)87–92, KGHRGF, with intermediate affinities (Kd ≈ 2 μM); and THPs including other collagenous sequences, or single-stranded sequences, negligibly (Kd ≫ 10 μM). Thus, heparin–type I collagen binding likely relies on an N-terminal basic triple-helical domain represented once within each monomer, and at multiple sites within fibrils. We next defined the features of type I collagen necessary for angiogenesis in a system in which type I collagen and heparin rapidly induce endothelial tube formation in vitro. When peptides, denatured or monomeric type I collagen, or type V collagen was substituted for type I collagen, no tubes formed. However, when peptides and type I collagen were tested together, only the most heparin-avid THPs inhibited tube formation, likely by influencing cell interactions with collagen–heparin complexes. Thus, induction of endothelial tube morphogenesis by type I collagen may depend upon its triple-helical and fibrillar conformations and on the N-terminal heparin-binding site identified here.

Mass spectrometric evidence for the enzymatic mechanism of the depolymerization of heparin-like glycosaminoglycans by heparinase II

Heparin-like glycosaminoglycans, acidic complex polysaccharides present on cell surfaces and in the extracellular matrix, regulate important physiological processes such as anticoagulation and angiogenesis. Heparin-like glycosaminoglycan degrading enzymes or heparinases are powerful tools that have enabled the elucidation of important biological properties of heparin-like glycosaminoglycans in vitro and in vivo. With an overall goal of developing an approach to sequence heparin-like glycosaminoglycans using the heparinases, we recently have elaborated a mass spectrometry methodology to elucidate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase I. In this study, we investigate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase II, which possesses the broadest known substrate specificity of the heparinases. We show here that heparinase II cleaves heparin-like glycosaminoglycans endolytically in a nonrandom manner. In addition, we show that heparinase II has two distinct active sites and provide evidence that one of the active sites is heparinase I-like, cleaving at hexosamine–sulfated iduronate linkages, whereas the other is presumably heparinase III-like, cleaving at hexosamine–glucuronate linkages. Elucidation of the mechanism of depolymerization of heparin-like glycosaminoglycans by the heparinases and mutant heparinases could pave the way to the development of much needed methods to sequence heparin-like glycosaminoglycans.

Mouse model of Sanfilippo syndrome type B produced by targeted disruption of the gene encoding α-N-acetylglucosaminidase

The Sanfilippo syndrome type B is an autosomal recessive disorder caused by mutation in the gene (NAGLU) encoding α-N-acetylglucosaminidase, a lysosomal enzyme required for the stepwise degradation of heparan sulfate. The most serious manifestations are profound mental retardation, intractable behavior problems, and death in the second decade. To generate a model for studies of pathophysiology and of potential therapy, we disrupted exon 6 of Naglu, the homologous mouse gene. Naglu−/− mice were healthy and fertile while young and could survive for 8–12 mo. They were totally deficient in α-N-acetylglucosaminidase and had massive accumulation of heparan sulfate in liver and kidney as well as secondary changes in activity of several other lysosomal enzymes in liver and brain and elevation of gangliosides GM2 and GM3 in brain. Vacuolation was seen in many cells, including macrophages, epithelial cells, and neurons, and became more prominent with age. Although most vacuoles contained finely granular material characteristic of glycosaminoglycan accumulation, large pleiomorphic inclusions were seen in some neurons and pericytes in the brain. Abnormal hypoactive behavior was manifested by 4.5-mo-old Naglu−/− mice in an open field test; the hyperactivity that is characteristic of affected children was not observed even in younger mice. In a Pavlovian fear conditioning test, the 4.5-mo-old mutant mice showed normal response to context, indicating intact hippocampal-dependent learning, but reduced response to a conditioning tone, perhaps attributable to hearing impairment. The phenotype of the α-N-acetylglucosaminidase-deficient mice is sufficiently similar to that of patients with the Sanfilippo syndrome type B to make these mice a good model for study of pathophysiology and for development of therapy.

Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: Direct evidence that lipoprotein lipase bridging occurs in vivo

Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis. In vitro studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs in vivo, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp156 to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7×; LPL1, 1.8×), core cholesteryl ether (LPL2, 2.3×; LPL1, 2.7×), and apolipoprotein (LPL1, 1.8×; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act in vivo to mediate VLDL removal from plasma and uptake into tissues in which it is expressed.

Complex Patterns of Alternative Splicing Mediate the Spatial and Temporal Distribution of Perlecan/UNC-52 in Caenorhabditis elegans

The unc-52 gene encodes the nematode homologue of mammalian perlecan, the major heparan sulfate proteoglycan of the extracellular matrix. This is a large complex protein with regions similar to low-density lipoprotein receptors, laminin, and neural cell adhesion molecules (NCAMs). In this study, we extend our earlier work and demonstrate that a number of complex isoforms of this protein are expressed through alternative splicing. We identified three major classes of perlecan isoforms: a short form lacking the NCAM region and the C-terminal agrin-like region; a medium form containing the NCAM region, but still lacking the agrin-like region; and a newly identified long form that contains all five domains present in mammalian perlecan.  Using region-specific antibodies and unc-52 mutants, we reveal a complex spatial and temporal expression pattern for these UNC-52 isoforms. As well, using a series of mutations affecting different regions and thus different isoforms of UNC-52, we demonstrate that the medium NCAM-containing isoforms are sufficient for myofilament lattice assembly in developing nematode body-wall muscle. Neither short isoforms nor isoforms containing the C-terminal agrin-like region are essential for sarcomere assembly or muscle cell attachment, and their role in development remains unclear.

Fibroblast growth factors 1 and 2 are distinct in oligomerization in the presence of heparin-like glycosaminoglycans

Fibroblast growth factor (FGF) 1 and FGF-2 are prototypic members of the FGF family, which to date comprises at least 18 members. Surprisingly, even though FGF-1 and FGF-2 share more than 80% sequence similarity and an identical structural fold, these two growth factors are biologically very different. FGF-1 and FGF-2 differ in their ability to bind isoforms of the FGF receptor family as well as the heparin-like glycosaminoglycan (HLGAG) component of proteoglycans on the cell surface to initiate signaling in different cell types. Herein, we provide evidence for one mechanism by which these two proteins could differ biologically. Previously, it has been noted that FGF-1 and FGF-2 can oligomerize in the presence of HLGAGs. Therefore, we investigated whether FGF-1 and FGF-2 oligomerize by the same mechanism or by a different one. Through a combination of matrix-assisted laser desorption ionization mass spectrometry and chemical crosslinking, we show here that, under identical conditions, FGF-1 and FGF-2 differ in the degree and kind of oligomerization. Furthermore, an extensive analysis of FGF-1 and FGF-2 uncomplexed and HLGAG complexed crystal structures enables us to readily explain why FGF-2 forms sequential oligomers whereas FGF-1 forms only dimers. FGF-2, which possesses an interface capable of protein association, forms a translationally related oligomer, whereas FGF-1, which does not have this interface, forms only a symmetrically related dimer. Taken together, these data show that FGF-1 and FGF-2, despite their sequence homology, differ in their mechanism of oligomerization.

Mutations in HYAL1, a member of a tandemly distributed multigene family encoding disparate hyaluronidase activities, cause a newly described lysosomal disorder, mucopolysaccharidosis IX

Hyaluronan (HA), a large glycosaminoglycan abundant in the extracellular matrix, is important in cell migration during embryonic development, cellular proliferation, and differentiation and has a structural role in connective tissues. The turnover of HA requires endoglycosidic breakdown by lysosomal hyaluronidase, and a congenital deficiency of hyaluronidase has been thought to be incompatible with life. However, a patient with a deficiency of serum hyaluronidase, now designated as mucopolysaccharidosis IX, was recently described. This patient had a surprisingly mild clinical phenotype, including notable periarticular soft tissue masses, mild short stature, an absence of neurological or visceral involvement, and histological and ultrastructural evidence of a lysosomal storage disease. To determine the molecular basis of mucopolysaccharidosis IX, we analyzed two candidate genes tandemly distributed on human chromosome 3p21.3 and encoding proteins with homology to a sperm protein with hyaluronidase activity. These genes, HYAL1 and HYAL2, encode two distinct lysosomal hyaluronidases with different substrate specificities. We identified two mutations in the HYAL1 alleles of the patient, a 1412G → A mutation that introduces a nonconservative amino acid substitution (Glu268Lys) in a putative active site residue and a complex intragenic rearrangement, 1361del37ins14, that results in a premature termination codon. We further show that these two hyaluronidase genes, as well as a third recently discovered adjacent hyaluronidase gene, HYAL3, have markedly different tissue expression patterns, consistent with differing roles in HA metabolism. These data provide an explanation for the unexpectedly mild phenotype in mucopolysaccharidosis IX and predict the existence of other hyaluronidase deficiency disorders.

Agrin in Alzheimer’s disease: Altered solubility and abnormal distribution within microvasculature and brain parenchyma

Agrin is a heparan sulfate proteoglycan that is widely expressed in neurons and microvascular basal lamina in the rodent and avian central nervous system. Agrin induces the differentiation of nerve-muscle synapses, but its function in either normal or diseased brains is not known. Alzheimer’s disease (AD) is characterized by loss of synapses, changes in microvascular architecture, and formation of neurofibrillary tangles and senile plaques. Here we have asked whether AD causes changes in the distribution and biochemical properties of agrin. Immunostaining of normal, aged human central nervous system revealed that agrin is expressed in neurons in multiple brain areas. Robust agrin immunoreactivity was observed uniformly in the microvascular basal lamina. In AD brains, agrin is highly concentrated in both diffuse and neuritic plaques as well as neurofibrillary tangles; neuronal expression of agrin also was observed. Furthermore, patients with AD had microvascular alterations characterized by thinning and fragmentation of the basal lamina. Detergent extraction and Western blotting showed that virtually all the agrin in normal brain is soluble in 1% SDS. In contrast, a large fraction of the agrin in AD brains is insoluble under these conditions, suggesting that it is tightly associated with β-amyloid. Together, these data indicate that the agrin abnormalities observed in AD are closely linked to β-amyloid deposition. These observations suggest that altered agrin expression in the microvasculature and the brain parenchyma contribute to the pathogenesis of AD.

Role of cysteines in Plasmodium falciparum circumsporozoite protein: Interactions with heparin can rejuvenate inactive protein mutants

Various pathogenic bacteria, viruses, and protozoan bind to glycosaminoglycan-based receptors on host cells and initiate an infection. Sporozoites of Plasmodium predominantly express circumsporozoite (CS) protein on their surface, which binds to heparan sulfate proteoglycans on liver cell surface that subsequently leads to malaria. Here we show that the interaction of free heparin with this parasite ligand has the potential to be a critical component of invasion. CS protein of P. falciparum contains four cysteines at positions 361, 365, 396, and 401. In this study, all four cysteine residues were mutagenized to alanine both individually and in different combinations. Conversion of cysteine 396 to alanine (protein CS3) led to a 10-fold increase in the binding activity of the protein to HepG2 cells. Replacement of cysteines at positions 361, 365, and 401 either alone or in different combinations led to a near total loss of binding. Surprisingly, activity in these inactive mutants could be effectively restored in the presence of submolar concentrations of heparin. Heparin also up-regulated binding of CS3 at submolar concentrations with respect to the protein but down-regulated binding when present in excess. Given the significantly different concentrations of heparin in different organs of the host and the in vitro results described here one can consider in vivo ramifications of this phenomenon for pathogen targeting of specific organs and for the functional effects of antigenic variation on receptor ligand interaction.