Question: Based on the article below,write a review of biological functions asso
ID: 210463 • Letter: Q
Question
Question: Based on the article below,write a review of biological functions associated with proteoglycans.
(THE FIGURES WERE NOT PROVIDED!!)
Title: Proteoglycans Form and Function
Introduction
It has been nearly 20 years since the original publication of a comprehensive classification of proteoglycan gene families [1]. For the most part, these classes have been widely accepted. However, a broad and current taxonomy of the various proteoglycan gene families and their products is not available. In contrast to the classification of glycosaminoglycans (GAGs), primarily based on the chemical structure of their repeating disaccharide units, classifying proteoglycans is a much more complex task [2]. We propose a comprehensive and simplified nomenclature of proteoglycans based on three criteria including: Cellular and subcellular location, overall gene/protein homology, and the presence of specific protein modules within their respective protein cores. Whereas the first two attributes have been utilized in the past for various nomenclatures, the third attribute is ofmore recent development and represents a sort of “intrinsic signature” for various protein cores. Indeed, modular design is based on the simple concept that protein cores are made up of finite units, like pieces of Lego. The units represent a minimum level of organization and a module can be thought of as a functional domain that affects cell–matrix dynamics. Another key feature is that each module/functional unit can be stable and can fold on its own, without being part of the large precursor protein. Thus, a module is a self-contained component. An example of this is the LG3 domain of endorepellin, the C-terminal globular-like domain of perlecan, which has recently been crystallized [3]. Below, we will critically assess the field of proteoglycans which now encompass forty three distinct genes and a much higher number of proteoglycans due to alternative splicing, thereby providing a very rich and biologically-active group of molecules. As hyaluronan and the enzymes involved in the synthesis and degradation of various GAGs are not covered in this review, readers are referred to recent reviews covering these closely-related subjects General features Four major proteoglycan classes encompass nearly all the known proteoglycans of the mammalian genome (Fig. 1). Observing the types of proteoglycans based on cellular and subcellular localization, we can see that there is only one intracellular proteoglycan, serglycin. This unique proteoglycan forms a class on its own as it is the only proteoglycan that carries heparin side chains. Serglycin is packaged in the granules of mast cells and serves as biological glue for most of the intracellular proteases stored within the granules [19] . Another general observation is that heparan sulfate proteoglycans (HSPGs) are prevalently associated with the cell surface or the pericellular matrix. The HSPGs are intimately associated with the plasmamembranes of cells, either directly via an intercalated protein core or via a glycosyl-phosphatidyl- inositol (GPI) anchor, and function as major biological modifiers of growth factors such as FGF, VEGF and PDGF among others. Similar functions are also performed by the HSPGs located in the basement membrane zone, in addition to their ability to interact with each other and with key constituents of the basement membrane, including various laminins, collagen type IV, and nidogen. Presentation of growth factors to their cognate receptors in a biologically-favorable form is a major function of cell surface and pericellular HSPGs. Another key role is participating in the generation and long range maintenance of gradients for morphogens during embryogenesis and regenerative processes. As we move away from the cells in a centrifugal manner, chondroitin- and dermatan sulfate-containing proteoglycans (CSPGs and DSPGs, respectively) predominate. These proteoglycans function as structural constituents of complex matrices such as cartilage, brain, intervertebral discs, tendons and corneas. Thus, among other functions, they provide viscoelastic properties, retain water and keep osmotic pressure, dictate proper collagen organization and are the main molecules responsible for corneal transparency. The extracellular matrix also contains the largest class of proteoglycans, the so-called small leucine-rich proteoglycans (SLRPs) which are themost abundant products in terms of gene number. These SLRPs can function both as structural constituent and as signaling molecules, especially when tissues are remodeled during cancer, diabetes, inflammation and atherosclerosis. SLRPs interact with several receptor tyrosine kinases (RTKs) and Toll-like receptors, thereby regulating fundamental processes including migration, proliferation, innate immunity, apoptosis, autophagy and angiogenesis. Below we will discuss the rationale for grouping certain proteoglycans in the same class and their overall biological function. Intracellular proteoglycans It is quite amazing that since the original cloning of serglycin, the first proteoglycan-encoding gene to be sequenced, no other true intracellular proteoglycan has been discovered. Serglycin occupies a class of its own insofar as it is the only proteoglycan that is covalently substituted with heparin due to its consecutive (and quite unique) Ser-Gly repeats, essentially a silk-like sequence. Serglycin has been utilized primarily by mast cells for the proper assembly and packaging of the numerous proteases that are released upon inflammation [19] . The defects in the formation of mast cell granules observed in Srgn/ mice are remarkably similar to those observed in mast cells derived from mice lacking N-deacetylase/N-sulfotransferase 2, a key enzyme involved in the sulfation of heparin [19] . Thus, serglycin promotes granular storage via electrostatic interaction between its highly-anionic heparin chains and basic residues within the various proteases of the secretory granules. It is becoming evident, however, that all inflammatory cells express serglycin and store it within intracytoplasmic granules where, in addition to proteases, serglycin binds and modulates the bioactivity of several inflammatory mediators, chemokines, cytokines and growth factors [20] . More recently, serglycin has been found in a wide variety of non-immune cells such as endothelial cells, chondrocytes and smooth muscle cells [21] . Cell-surface serglycin promotes adhesion of myeloma cells to collagen I and affects the expression ofMMPs [22] . These findings have been corroborated by in vivo studies where serglycin knockdown attenuates the multiple myeloma growth in immunocompromised mice [23] . It has been proposed that some of these effects are mediated by a specific interaction between serglycin and cell-surface CD44 [23] , a known receptor for hyaluronan [24,25] . It has been recently shown that serglycin is a key component of the cell inflammatory response in activated primary human endothelial cells as both LPS and IL-1 increase its synthesis and secretion [26] . Notably, serglycin can be substituted with chondroitin sulfate (CS), and in several circulating cells serglycin contains lower sulfated CS-4 chains [21] . In contrast, several hematopoietic cells (mucosal mast cells, macrophages etc.) express serglycin with highly sulfated CS-E. Although the significance of this phenomenon is not fully appreciated, it is likely that these isoforms of serglycin might have different functions in a cell-context specific manner. Serglycin is a marker of immature myeloid cells and interacts with many bioactive components including histamine, TNF- and proteases [27] . In general, serglycin expression correlates with a more aggressive malignant phenotype and it has been recently proposed that serglycin protects breast cancer cells from complement attack, thereby supporting cancer cell survival and progression [28] . Cell surface proteoglycans In this class, there are thirteen genes, seven encoding transmembrane proteoglycans and six encoding GPI-anchored proteoglycans. With the exception of two gene products, NG2 and phosphacan, all contain heparan sulfate side chains. Syndecans The eponym syndecan was coined by the late Merton Bernfield [29] to define a class of transmembrane proteoglycans that would connect (from the Greek syndein, “bind together”) the surface of the cells to the underlying extracellular matrix. The syndecan family now comprises four distinct genes encoding single-pass transmembrane protein cores which include an ectodomain, a transmembrane region and an intracellular domain [4,30] (Fig. 2). The ectodomains exhibit the lowest amount of amino acid sequence conservation, no more than 10–20%, in contrast to the transmembrane and cytoplasmic domains which are 60–70% identical. A recent study has shown that the ectodomain of syndecans is natively disordered and this characteristic allows syndecans to interact with a variety of proteins and ligands, thereby providing enrichment in their biological function [31]. The ectodomain contains the GAG attachment sites, which are often covalently-linked to HS and sometimes to CS, making syndecans hybrid proteoglycans. Several cell types shed syndecan into the pericellular environment through the action of MMPs. For example, it has recently been shown that shed syndecan-2 retards angiogenesis by inhibiting endothelial cell migration [32], a key step in neovascularization [33]. The transmembrane domain contains a dimerization motif (GxxxG) that mediates both homo-dimerization and hetero-dimerization [30]. The intracellular domain is composed of two regions of conserved amino acid sequence (C1 and C2), separated by a central variable sequence of amino acids that is distinct for each family member (V) [34]. Notably, the C-terminus of all the four syndecans harbors a unique signature (EFYA) that binds PDZ-containing proteins. Generally, PDZ-containing proteins contribute to a proper anchor of transmembrane proteins to the cytoskeleton, thereby holding together large signaling complexes. Syndecans are involved in a wide variety of biological functions, too vast to be reviewed here, but reviewed recently [5,30,34]. Briefly, syndecans bind numerous growth factors, especially through their HS chains, and dictate morphogen gradients during development. In concert with other cellsurface HSPGs, syndecans can act as endocytosis receptors and are also involved in the uptake of exosomes [35]. Syndecans play key roles as co-receptors for many RTKs and can also function as receptors for atherogenic lipoproteins [36]. Indeed, there is strong genetic evidence that syndecan-1 is the main HSPG mediating clearance of triglyceride-rich lipoproteins derived from either the liver or from intestinal absorption [37]. Many, if not all the syndecans, can also act as soluble HSPGs via partial proteolysis of their juxtamembrane region releasing their whole ectodomains. This shedding is considered a powerful post-translational modification that can regulate the amount of HSPG linked to the cell surface and that present in the pericellular microenvironment [30]. Several inflammatory cytokines can induce syndecan shedding by triggering outside-in signaling and by activating several metalloproteinases. In the case of hepatocytes, shedding of syndecan-1 occurs via PKC-dependent activation of ADAM17, and this impairs VLDL catabolism and promotes hypertriglyceridemia [38] . Importantly, soluble syndecan-1 promotes the growth of myeloma tumors in vivo [39] , and this process, i.e. the shedding of syndecan-1, is enhanced by heparanase [40] , thereby offering a novel mechanism for promoting cancer growth and metastasis [41,42] . Notably, chemotherapy stimulates syndecan-1 shedding, a potential drawback of the treatment that could potentially favor tumor progression [43] . The biological interplay between heparanase- evoked shedding of syndecan-1 and myeloma cells leads to enhanced angiogenesis [44] , further supporting cancer growth. As mentioned above, however, shed syndecan-2 inhibits angiogenesis via a paracrine interaction with the protein tyrosine phosphatase receptor CD148, which in turn deactivates 1-containing integrins [32] , presumably 1 1 and 2 1, two main angiogenesis receptors. In contrast, the ortholog syndecan-2 is required for angiogenic sprouting during zebrafish development [45] . An emerging new role for syndecan-1 is linked to its ability to reach the nuclei in a variety of cells. Initial observations showed that myeloma and mesothelioma cells contain syndecan-1 in their nuclei [46,47] and this nuclear translocation is also regulated by heparanase [46] , indicating that there must be a cellular receptor for shed syndecan-1 that could mediate its nuclear targeting and transport. In support of these studies are previous observations that exogenous HS can translocate to the nuclei and modulate the activity of DNA Topoisomerase I [48] and histone acetyl transferase (HAT) [49] . N-terminal acetylation of histones by HAT is linked to transcriptional activation, and this process is finely tuned by its counteracting enzyme, histone deacetylase (HDAC). Heparanase-evoked loss of nuclear syndecan-1 causes an increase in HAT enzymatic activity and enhances transcription of pro-tumorigenic genes [50] . Syndecan-1 that is shed from myeloma tumor cells is uptaken by bone marrow stromal cells and is transported to the nuclei by amechanismthat requires its HS chains, as this process is inhibited by heparin and chlorate [51] . Once nuclear, soluble syndecan-1 binds to HAT p300 and inhibits its activity, thereby providing a new mechanism for tumor– host cell interaction and cross-talk [52] . CSPG4/NG2 The melanoma-associated chondroitin sulfate proteoglycan (MCSP) was discovered over 30 years ago as a transmembrane proteoglycan and a highly immunogenic tumor antigen ofmelanoma tumor cells. This proteoglycan has been subsequently detected in various species, with many names designating the same gene product. The rat ortholog of MCSP is called nerve/glial antigen 2 (NG2) [53] , while the term CSPG4 designates the human gene. We will use CSPG4/NG2 terminology with the idea that some of the functional properties have not been fully described in the human and rat species [54] . CSPG4/NG2 is a single-pass, type I transmembrane proteoglycan carrying one chondroitin sulfate chain, and harboring a large ectodomain composed of three subdomains (Fig. 2 ). The N-terminal domain (D1 subdomain) contains two laminin-like globular (LG) repeats. It is likely that the LG domains as in other proteoglycans (i.e. perlecan and agrin, see below) mediate ligand binding, cell– matrix and cell– cell interactions, as well as interaction with integrins and receptor tyrosine kinase (RTK). The central subdomain D2 contains 15 tandem repeats of a new module called CSPG [54] . The CSPG repeat is a cadherin-like and tumorrelevant module which is predicted to be involved in cell– matrix interaction, further modulated by the CS chain covalently attached to this module. Indeed, CSPG modules bind to collagens V and VI, FGF and PDGF. The juxtamembrane subdomain D3 contains a carbohydrate modification able to bind integrins and galectin, as well as numerous protease cleavage sites. Accordingly, the intact ectodomain and fragments thereof can be detected in sera from normal and melanoma-carrying patients [54] . The transmembrane domain of CSPG4/NG2 is quite interesting insofar as it has a unique Cys residue, generally not found in transmembrane regions. The intracellular domain harbors a proximal region with numerous Thr phospho-acceptor sites for PKC and ERK1/2, and a distal region encompassing a PDZ-binding module similar to the syndecan family. The latter can bind to the PDZ domain of several scaffold proteins involved in intracellular signaling, including syntenin, MUPP1 and GRIP1. Functionally, CSPG4/NG2 proteoglycan promotes tumor vascularization [55] and because of its predominant perivascular localization, CSPG4/NG2 may modulate the availability of FGF at the cell surface as well as the bioactivity and signal transduction of FGF receptors [56] . This CSPG binds to collagen VI in the tumor microenvironment and promotes cell survival and adhesion via the PI3K pathway [57] . Indeed, targeting CSPG4/NG2 in two animal models of highly-malignant brain tumors reduces tumor growth and angiogenesis [58] . Moreover, a combinatorial treatment using activated natural killer cells and a monoclonal antibody toward CSPG4/NG2 is capable of eradicating glioblastoma xenografts more efficiently than single therapies [59] . It has recently been discovered that NG2 controls the directional migration of oligodendrocyte precursor cells by constitutively stimulating RhoA GTPases [60]. Based on NG2 ability to regulate adhesion RhoA GTPase and growth factor activities, it is likely that this transmembrane proteoglycan might play a key role in regulating cell polarity in response to extracellular cues [61] . Perdido/Kon-tiki , the Drosophila ortholog of mammalian CSPG4 , genetically interacts with integrins during Drosophila embryogenesis, and its loss is embryonic lethal [62] . RNAi-mediated suppression of Perdido/Kon-tiki in the muscles, just before adult myogenesis starts, induces misorientation and detachment of Drosophila adult abdominal muscle, generating a phenotype similar to the embryonic lethal ones [63] . Thus, it is possible that, based on its high conservation through species, mammalian CSPG4 could also play a role in myogenesis and function as well. A recent study has added another function to CSPG4 by involving this cell surface proteoglycan in the pathogenesis of severe pseudomembranous colitis. CSPG4 acts as a receptor for the Clostridium difficile toxin B, one of the key toxins secreted by this gram-positive and spore-forming anaerobic bacillus [64] . The interaction occurs between the N-terminus of CSPG4 and the C-terminus of toxin B. This discovery, if confirmed in future studies, opens new therapeutic targets for the treatment of this severe and often lethal form of enterocolitis. Betaglycan/TGF type III receptor In 1991, two back-to-back papers reported on the isolation and cloning of a membrane-anchored proteoglycan with high affinity for TGF , and thus named betaglycan [65,66] . Betaglycan, also known as TGF type III receptor (TGFB3), is a single-pass transmembrane proteoglycan that belongs to the TGF superfamily of co-receptors (Fig. 2 ). The extracellular domain contains several potential GAG attachment sites and protease-sensitive sequences near the plasma membrane. The short intracellular domain is highly enriched in Ser/Thr (N 40%) and some of these residues are candidate sites for PKC-mediated phosphorylation [65] . Betaglycan amino acid sequence is highly similar to that of endoglin, a close member of the same superfamily. The membrane-proximal ectodomain of betaglycan contains a unique module called zona pellucida (ZP)-C [67] . The ZP module is a structural element typically found in the ectodomain of eukaryotic proteins composed of a Cys-rich bipartite structure joined by a linker. Generally, proteins harboring ZP modules tend to polymerize and assemble into long fibrils of specialized extracellular matrices [67] . In the case of betaglycan and endoglin these ZP modules are not utilized for polymerization, rather they function as membrane co-receptors for the TGF superfamily members [68] . The intracellular domain contains a PDZ-binding element similar to that observed in the syndecan family of proteoglycans (Fig. 1 ). Betaglycan is a ubiquitously-expressed cell surface proteoglycan that acts as a co-receptor for members of the TGF superfamily of Cys knot growth factors which also include activins, inhibins, GDFs and BMPs [69,70] . For example, betaglycan enhances the binding of all the TGF isoforms to the signaling TGF complex [71] and is needed for TGF 2 high-affinity interaction with the receptor complex. Betaglycan also blocks the aggressiveness of ovarian granulosa cell tumors by suppressing NF- B-evoked MMP2 expression [72] . Betaglycan, together with other TGF -binding SLRPs, i.e. decorin and biglycan (see below), can be cleaved by granzyme B, thereby releasing an active form of TGF [73] . Ectodomain shedding of betaglycan is indeed necessary for betaglycanmediated suppression of TGF signaling and breast cancer migration and invasion [74] . The ability of betaglycan to affect epithelial mesenchymal transformation [70] , together with genetic evidence of embryonic lethality in Tgfbr3/ mice, suggests that betaglycan may play a unique and non-redundant function during development. Another important feature of betaglycan is its ability to modulate the subcellular topology of the signaling receptor complex via its PDZ-binding domain, which interacts with PDZ-containing proteins such as -arrestin [75] . This interaction, as well as that between betaglycan intracellular domain and GIPC, would stabilize betaglycan at the cell surface and potentiate its bioactivity. Finally, betaglycan is involved in regulating many functions including reproduction and fetal growth [75] , and is a putative tumor suppressor in many forms of cancer [76] . Several additional betaglycan-evoked activities have been recently reviewed elsewhere [75] . Phosphacan/receptor-type protein tyrosine phosphatase Phosphacan, originally isolated from rat brain, is a CSPG that interacts with neurons and neural cell-adhesion molecules (N-CAM) and corresponds to the soluble ectodomain of a Receptor-type protein tyrosine phosphatase (RPTP ) [77] . The phosphacan gene (PTPRZ1 ) encodes a single-pass type I membrane protein with a relatively large ectodomain harboring an N-terminal module homologous to the alpha-carbonic anhydrase (Fig. 2 ). Distal to this, there is a fibronectin type III domain. The ectodomain contains six Ser-Gly repeats, at least four of which are flanked by acidic residues suggesting potential glycanation sites. Sporadically, phosphacan can also be substituted with keratan sulfate chains. Notably, alternative splice variants encoding different protein isoforms have been described but their full-length nature has not yet been established. Functionally, the ectodomain of phosphacan mediates cell– cell adhesion by hemophilic binding. In addition, phosphacan's ability to bind N-CAM and tenascin in a calcium-dependent manner suggests that RPTPs may also modulate cellular interactions via heterophilic mechanisms [77] . Indeed, phosphacan blocks the growth-promoting ability of N-CAM, axonin-1 TAG-1 and tenascin, and is crucial in the oriented movement of post-mitotic cells during cortical development of the brain [78] .Moreover, phosphacan binds contactin, another member of the Ig superfamily like N-CAM, and the extracellular portion of the voltage-gated sodium channel [79] . The latter interaction appears to be mediated by the carbonic anhydrase-like module of phosphacan's ectodomain. It has been proposed that phosphacan, as an integral extracellular matrix constituent of the neural stem cell compartment, would contribute to the privileged microenvironment that supports self-renewal and maintenance of the neural stem cell niche [80] . Glypicans/GPI-anchored proteoglycans Glypicans (GPC) are HSPGs that are bound to the plasma membrane via a C-terminal lipid moiety known as GPI, for glycosylphosphatidylinositol, linkage or anchor (Fig. 2 ). There are six independent genes in the mammalian genome which can be subdivided into two broad classes: GPC1/2/3/6 and GPC3/5 with orthologs present across Metazoan including Dally and Dlp in Drosophila melanogaster [81] . Although most of the protein core is unique to this family, there is a stretch of amino acid in the ectodomain of the protein core with similarity to the Cys-rich domain of Frizzled proteins. There are two unique features in the structural organization of all glypicans, with potentially important functional implications. First and in contrast to syndecans, the attachment of the GAG chains – mostly HS chains – is located near the juxtamembrane region. This allows the three linear HS chains to span a great deal of plasma membrane surface, thereby presenting various morphogens and growth factors in an active configuration to their cognate receptors. Indeed, glypicans bind to and modulate the activity of Hedgehog (Hh), Wnt, and FGFs [82–84] . More recently, it has been shown that glypican-3 binds to Frizzled thereby acting directly in the modulation of canonical Wnt signaling [85] . Second, glypicans are dually processed via partial proteases and lipases. In the former case, the ectodomain of glypicans is processed via endoproteolytic cleavage by a furin-like convertase. This processing generates two subunits that are then bound via disulfide bonds, in a way similar to the Met receptor. In the latter case, the entire glypican proteoglycan is released from the plasma membrane via an extracellular lipase (Notum ) that cleaves the GPI anchor. Drosophila studies have shown that the Notum -mediated release of glypican can regulate morphogen gradients including Wnt, BMP and Hh gradients [84] . Notably, the anchorless GPC-1, devoid of the GPI anchor, is a stable -helical protein that rests high concentrations of urea and guanidine HCL [86] . Unfolding data are consistent with a two-state model, suggesting that GPC-1 protein core is a densely-packed globular protein. In agreement with these data, the crystal structure of the Drosophila glypican Dally-like protein has revealed an extended -helical fold [87] . The crystal structure of human GPC-1 is very similar to Drosophila Dally-like , and consists of a stable -helical domain with 14 conserved Cys residues, followed by a GAG attachment site that is exclusively substituted with HS chains [88] . Of interest, removal of the -helical domain leads to substitution with CS chains instead of HS chains, indicating that there is a “ message” embedded in the -helical domain that drives a different posttranslational modification [88] . Functionally, glypicans have been involved in the control of tumor growth and angiogenesis. For example, glypican-3 has been implicated in cancer and growth control. Human mutations of GPC3 cause the rare X-linked Sympson–Golabi–Behmel (SGB) syndrome, characterized by both pre- and post-natal overgrowth, abnormal craniofacial features, cardiovascular anomalies, renal dysplasia and urinary tract malformations [84] . Originally, it was hypothesized that GPC3 was an inhibitor of IGF-II, given the prominent function of IGF-II in developmental growth. However, it was later found that the levels of IGF-II do not change in Gpc3/ mice nor does GPC3 interacts with IGF-II. It appears that GPC3 is an inhibitor of the Hh signaling, insofar as the Hh-dependent signaling activity is elevated in Gpc3/ mice. Moreover, purified glypican-3 binds with high affinity to Indian and Sonic Hh as well as it competes with Patched for Hh binding [83,89] . A recent study has shown that processing by convertases is required for GPC3-evoked suppression of Hh signaling, and this process is dependent on the HS chains and their degree of sulfation [90] . Thus, the glypican family is not only complex in nature, but is also the control of various modifying enzymes (proteases and lipases) that modulate its biological activity. We are positive than many “ surprises” will happen in the future regarding unsuspected biological functions of various glypicans. Pericellular and basement membrane zone Proteoglycans. This group of four proteoglycans is closely associated with the surface of many cell types The key for the various modules is provided in the bottom panel. anchored via integrins or other receptors, but they can also be a part of most basement membranes. Pericellular proteoglycans are mostly HSPGs and include perlecan and agrin, which share homology especially at their C-termini, and collagens XVIII and XV, which share homology at their N- and C-terminal noncollagenous domains (Fig. 1 ). Perlecan Perlecan is a modular HSPG encoded by a large gene [91,92] with a complex promoter [93–95] . The ~500-kDa protein core is composed of 5 domains with homology to SEA, N-CAM, IgG, LDL receptor and laminin [96,97] (Fig. 3 ). The terminal LG3 domain has been crystallized and reveals a jellyroll fold characteristic of other LG modules [3] . Perlecan is expressed by both vascular and avascular tissues [97–101] , and is ubiquitously located at the apical cell surface [102,103] and basement membranes [98,104–106] . Perlecan regulates various biological processes primarily because of its widespread distribution [101,105] and its ability to interact with various ligands and RTKs [107] , and more recently the potential utilization of perlecan splice variants in mast cells [108] . Perlecan is an early responsive gene and is induced by TGF [109] and repressed by interferon [95] . The heparan sulfate chains of perlecan and the protein core can be cleaved by heparanase and various proteases [110–112] , respectively, releasing various pro-angiogenic Perlecan is involved in modulating cell adhesion [114,115] , lipid metabolism [116] , thrombosis and cell death [117,118] , biomechanics of blood vessels and cartilage [119–121] , skin and endochondral bone formation [122,123] , and osteophyte formation [124] . Perlecan binds and modulates the activity of several growth factors and morphogens [106,125–129] and its expression is often deregulated in several types of cancer [130–134] . In Drosophila , perlecan, known as Trol (for terribly reduced optical lobe) regulates Fgf and Hh signaling to activate neural stem signaling [135,136] . In addition, Trol is essential for the architecture and maintenance of the lymph gland and for the proliferation of blood progenitor cells [137] . Loss of Trol is associated with premature differentiation of hemocytes and this phenotype can be rescued by ectopic expression of Hh [137] . In mice, Hspg2 controls neurogenesis in the developing telencephalon [138] . Moreover, perlecan can act as a lipoprotein receptor and mediate its endocytosis and catabolism [116] . Specifically, domain II of perlecan has been shown to bind low density lipoproteins and this interaction is mediated by the O-linked oligosaccharides [139] , suggesting an important role for perlecan in atherogenesis and lipid retention. Perlecan is a complex regulator of vascular biology and tumor angiogenesis [33,140,141] by performing a dual function: via the N-terminal HS chains, perlecan is pro-angiogenic [96] by binding and presenting VEGFA and various FGFs to their cognate receptors [33,141–152] . Moreover, heparanase- mediated cleavage of basement membrane perlecan releases FGF10 and enhances salivary gland branching morphogenesis [153] . Indeed, ablating Hspg2 or preventing Hspg2 expression in early embryogenesis causes severe cardiovascular defects [154–157] . The critical role for the N-terminal HS chains of perlecan has been elegantly demonstrated by the generation of mice harboring a genomic deletion of exon 3, designated Hspg 23/3 mice, which encodes the SGDs responsible for the covalent attachment of HS chains [158] . These mutant mice have impaired angiogenesis, delayed healing after experimental wounding and suppression of tumor growth [159] . When challenged with flow cessation of the carotid artery, the Hspg 23/3 mice show an enhanced intimal hyperplasia and smooth muscle cell proliferation [160,161] . Moreover, during mouse hind-limb ischemia, the HS chains of perlecan are key regulators of the angiogenic response [162] .Collectively, these studies reaffirmthe role of HS perlecan in modulating pro-angiogenic factors such as FGF2, VEGFA and PDGF. More recently other functions of perlecan have been discovered. Using a lethality-rescued Hspg2/ where perlecan was reintroduced into the cartilage, it was found that perlecan deficiency leads to significant depression of endothelial nitric oxide synthase [163] . This leads to endothelial cell dysfunction, as shown by attenuated endothelial relaxation, likely as a consequence of endothelial nitric oxide synthase expression. This is another example of how a secreted HSPG affects the biology of vascular endothelial cells likely through a receptor-mediated signaling pathway. Another recently unveiled function of perlecan is its ability to bind the clustering molecule gliomedin [164] . In this case, perlecan binds dystroglycan at nodes of Ranvier which are required for fast conduction and accumulation of Na+ channels. Perlecan seems to enhance clustering of nodes of Ranvier components via a specific interaction with gliomedin. Thus, perlecan may have specific roles in the biology and pathophysiology of peripheral nodes [164] . In contrast to the pro-angiogenic N-terminal domain I, the C-terminal processed form of perlecan domain V, named endorepellin [165] , has a nearly opposite function: it inhibits endothelial cell migration, capillary morphogenesis, and in vivo angiogenesis [166–169] . A global proteomic analysis of human serum has identified endorepellin as a major circulating protein [170] . Moreover, endorepellin has been detected in extracts of fetal cartilage, exclusively in the hypertrophic zone, and it was speculated that processing of perlecan protein core in the growth plate could play a role in inhibiting blood vessel invasion or formation in cartilage [171] . Moreover, MMP-7 processing of perlecan in the prostate cancer stroma acts as a molecular switch to favor cancer invasion [112] . Thus, processed forms of perlecan protein core harboring domains III and IV can function as protumorigenic factors. Endorepellin binds to the 2 1 integrin receptor [140,166,194] , and tumor xenografts generated in 21/ mice are insensitive to systemic delivery of endorepellin [168] . Endorepellin triggers the activation of the tyrosine phosphatase SHP-1 which, in turn, dephosphorylates and inactivates various RTKs including VEGFR2 [195] . Soluble endorepellin alters the proteomic profile of human endothelial cells [196] , and exerts a dual receptor antagonism by concurrently targeting VEGFR2 and the 2 1 integrin [197] . Notably, the proximal LG1/2 domains bind the Ig3– 5 domain of VEGFR2 while the terminal LG3 domain, release by BMP-1/Tolloid-like metalloproteinases [174] , binds the 2 1 integrin [198] . This dual signaling causes: (a) Disassembly of actin filaments and focal adhesions, via the 2 1 integrin, leading to suppression of endothelial cell migration [198,199] , and (b) Activation of SHP-1 dephosphorylates Tyr1175 , a key residue in the cytoplasmic tail of VEGFR2, and consequent transcriptional inhibition of VEGFA [200] . More recently, we have discovered that endorepellin induces autophagy in endothelial cells via VEGFR2 signaling [201] , similar to decorin (see below). This novel function could contribute to the angiostatic properties of this interesting fragment of perlecan protein core. Agrin The second pericellular/basement membrane HSPG is agrin. A C-terminal portion of agrin lacking HS chains was first isolated from the Torpedo electric organ as an agent responsible for acetylcholine receptor (AChR) clustering, thereby the eponym agrin, from the Greek ageirein , meaning “ to assemble” [202] . The majority of the research on agrin in mammalians has focused on agrin's contribution to the control of the postsynaptic apparatus in the neuromuscular junction. However, after many years of research, it was serendipitously discovered that agrin was indeed anHSPGinteracting with N-CAM in the avian brain [203] . Subsequently, orthologs of agrin have been cloned from multiple species and are all highly homologous. Agrin has a multimodular structural organization that is homologous to that of perlecan with potential generation of several splice isoforms. In addition to secreted full-length brevican, an isoform of brevican encoded by a shorter 3.3 kb mRNA and highly expressed during post-natal development, is linked to the plasma membrane via a GPI anchor [273] . Notably, the GPI-anchored brevican lacks EGF, C-type lectin and CRP modules but contains a stretch of hydrophobic amino acids resembling the GPI-anchor. Brevican is located at the outer surface of neurons and is enriched at perisynaptic sites. Brevican interacts with tenascin- R and fibulin-2 via its G3-like domain [274] . Functionally, brevican has been implicated in glioma tumorigenesis, nervous tissue injury and repair, and in Alzheimer's disease [274] . However, many more studies need to be performed before a clear picture of brevican's biology can be clearly drawn. Final considerations Of the 43 genes encoding full-time proteoglycans, only 33 appear to be glycanated. Thus, roughly 1 in 10,000 genes in the human genome codes for a proteoglycan protein core. This is quite amazing and indicates that proteoglycans play fundamental and often vital functions necessary for life to operate and evolve.We are confident that new proteoglycans will be discovered in the future. One of the major difficulties in finding new proteoglycans is their large size and negative charge. Both hinder proper separation in conventional acrylamide or 2D gels used for routine proteomic studies of various biological fluids and tissues. However, as in the case of agrin and collagen XVIII which were studied for several years without knowing their proteoglycan nature, it is likely that there will be significant discoveries of known proteins as being members of the “restricted” proteoglycan gene family. We hope that this nomenclature will help researchers who want to familiarize themselves with our exciting and growing field of proteoglycan biology.
Explanation / Answer
Review:
Proteoglycan gene families:
A comprehensive nomenclature of proteoglycans is based on three criteriae that includes:
The third one is more recent and represents “Intrinsic Signature” for different protein cores.
Protein cores are made up of finite units. These units represent a level of organization and can be thought as a functional domain that affects cell–matrix dynamics. Another feature is that each functional unit can be stable and can fold on its own.
Example :
Proteoglycans have forty three distinct genes and a higher number of proteoglycans because of alternative splicing. There are four major proteoglycan classes that encompass all the known proteoglycans of the mammalian genome.
Proteoglycans are also based on cellular and subcellular localization. There is only one intracellular proteoglycan i.e. Serglycin. Serglycin is present in the granules of mast cells and acts as biological glue for most of the intracellular proteases stored within the granules. Serglycin is the first proteoglycan-encoding gene to be sequenced. Serglycin occupies a class of its own as it is the only proteoglycan that is substituted with heparin due to its consecutive Ser-Gly repeats. Serglycin has been utilized by mast cells for the proper assembly and packaging of the numerous proteases that are released upon inflammation. Cell-surface serglycin promotes adhesion of myeloma cells to collagen I and affects the expression of MMPs. Several hematopoietic cells express Serglycin. Serglycin is a marker of immature myeloid cells and interacts with many bioactive components.
The HSPGs are associated with the plasma membranes of cells, either directly via an intercalated protein core or via a glycosyl-phosphatidyl- inositol (GPI) anchor. They are the major biological modifiers of growth factors such as FGF and VEGF.
Chondroitin and Dermatan are the sulfate-containing proteoglycans. These proteoglycans function as structural constituents of complex matrices such as cartilage, brain, intervertebral discs, tendons and corneas. They also provide viscoelastic properties and keep osmotic pressure, dictate proper collagen organization and are the main molecules responsible for corneal transparency.
Small leucine rich proteoglycans (SLRPs) are the most abundant products in terms of gene number. These can function both as structural constituent and as signaling molecules, when tissues are remodeled during cancer, diabetes, inflammation and atherosclerosis. SLRPs interact with receptor tyrosine kinases (RTKs) and Toll-like receptors which regulates fundamental processes including migration, proliferation, innate immunity, apoptosis, autophagy and angiogenesis.
Cell Surface Proteoglycans : There are thirteen genes, seven encodes transmembrane proteoglycans and six encodes GPI-anchored proteoglycans. All gene products contain heparan sulfate side chains except NG2 and phosphacan.
Syndecans are a class of transmembrane proteoglycans. It comprises of four distinct genes encoding transmembrane protein cores which include an ectodomain, a transmembrane region and an intracellular domain.
The ectodomain exhibit the lowest amount of amino acid sequence conservation. The ectodomain contains the GAG attachment sites, which are often covalently-linked to HS and when they linked to CS, forms syndecans hybrid proteoglycans. Syndecan-2 reduces angiogenesis by inhibiting endothelial cell migration.
The transmembrane domain contains a dimerization motif that mediates both homo-dimerization and hetero-dimerization. PDZ-containing proteins contribute to a proper anchor of transmembrane proteins to the cytoskeleton. The juxtamembrane subdomain contains a carbohydrate modification able to bind integrins and galectin, as well as numerous protease cleavage sites. The transmembrane domain of CSPG4/NG2 has a unique Cys residue, generally not found in transmembrane regions. The latter can bind to the PDZ domain of several scaffold proteins involved in intracellular signaling, including Syntenin, MUPP1 and GRIP1.
The intracellular domains have a proximal region with numerous Thr-phospho acceptor sites for PKC and ERK1/2, and a distal region encompassing a PDZ-binding module similar to the syndecan family. This CSPG binds to collagen VI in the tumor microenvironment and promotes cell survival and adhesion via the PI3K pathway. NG2 controls the directional migration of oligodendrocyte precursor cells by constitutively stimulating RhoA GTPases.
Betaglycan which is known as TGF type III receptor (TGFB3), is a single-pass trans membrane proteoglycan that belongs to the TGF superfamily of co-receptors. The membrane-proximal ectodomain of betaglycan contains a unique module called zona pellucida. The Zona Pellucida module is a structural element typically found in the ectodomain of eukaryotic proteins composed of a Cys-rich bipartite structure joined by a linker. Generally, proteins harboring Zona Pellucida modules tend to polymerize and assemble into long fibrils of specialized extracellular matrices. Betaglycan is a ubiquitously-expressed cell surface proteoglycan that acts as a co-receptor for members of the TGF superfamily of Cys knot growth factors which also include activins, inhibins, GDFs and BMPs. Betaglycan blocks the aggressiveness of ovarian granulosa cell tumors by suppressing MMP2 expression. Betaglycan modulate subcellular topology of the signaling receptor complex via PDZ-binding domain, which interacts with PDZ-containing proteins such as -arrestin . Betaglycan is involved in regulating many functions including reproduction and fetal growth and is a tumor suppressor in many forms of cancer.
Glypicans are HSPGs that bound to the plasma membrane via C-terminal lipid known as GPI (glycosylphosphatidylinositol). There are two unique features in the structural organization of all glypicans. First is the attachment of the GAG chains – mostly HS chains – is located near the juxta membrane region. This allows the three linear HS chains to span plasma membrane surface, thereby presenting various morphogens and growth factors in an active configuration to their cognate receptors. Glypicans bind to and modulate the activity of Hedgehog (Hh), Wnt, and FGFs. Glypicans have been involved in the control of tumor growth and angiogenesis. Glypican-3 has been implicated in cancer and growth control.
Pericellular proteoglycans are mostly HSPGs and include Perlecan and Agrin, which share homology at their C-termini and collagens which share homology at their N- and C-terminal non-collagenous domains. Perlecan is an early responsive gene and is induced by TGF and repressed by interferon .
The C-terminal processed form of perlecan domain V called endorepellin. It inhibits endothelial cell migration, capillary morphogenesis, and in vivo angiogenesis. Endorepellin has been detected in extracts of fetal cartilage. Endorepellin triggers the activation of the tyrosine phosphatase SHP-1 which then dephosphorylates and inactivates various RTKs. Endorepellin binds to the 2 1 integrin receptor. Soluble endorepellin alters proteomic profile of human endothelial cells and exerts dual receptor antagonism by targeting VEGFR2 and the 2 1 integrin.
The second pericellular membrane HSPG is Agrin. C-terminal portion of Agrin lacking HS chains was an agent responsible for acetylcholine receptor (AChR) clustering. Agrin is responsible for the control of the post synaptic apparatus in the neuromuscular junction. Agrin has a multi modular structural organization that is homologous to Perlecan with generation of several splice isoforms.
Brevican is located at the outer surface of neurons and mostly at perisynaptic sites. Brevican interacts with tenascin-R and fibulin-2 via its G3-like domain. Brevican has been implicated in glioma tumorigenesis, nervous tissue injury and repair in Alzheimer's disease.
43 genes encodes full-time proteoglycans and only 33 appear to be glycanated. Thus 1 in 10,000 genes in the human genome codes for a proteoglycan protein core. In the case of agrin and collagen XVIII, there will be significant discoveries of known proteins as being members of the “restricted” proteoglycan gene family. Proteoglycans play fundamental and vital functions necessary for life to operate.
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