Molecular mechanism of decision-making in glycosaminoglycan biosynthesis.

Two major glycosaminoglycan types, heparan sulfate (HS) and chondroitin sulfate (CS), control many aspects of development and physiology in a type-specific manner. HS and CS are attached to core proteins via a common linker tetrasaccharide, but differ in their polymer backbones. How core proteins are specifically modified with HS or CS has been an enduring mystery. By reconstituting glycosaminoglycan biosynthesis in vitro, we establish that the CS-initiating N-acetylgalactosaminyltransferase CSGALNACT2 modifies all glycopeptide substrates equally, whereas the HS-initiating N-acetylglucosaminyltransferase EXTL3 is selective. Structure-function analysis reveals that acidic residues in the glycopeptide substrate and a basic exosite in EXTL3 are critical for specifying HS biosynthesis. Linker phosphorylation by the xylose kinase FAM20B accelerates linker synthesis and initiation of both HS and CS, but has no effect on the subsequent polymerisation of the backbone. Our results demonstrate that modification with CS occurs by default and must be overridden by EXTL3 to produce HS.

Structure of the transmembrane protein 2 (TMEM2) ectodomain and its apparent lack of hyaluronidase activity.

Background: Hyaluronic acid (HA) is a major polysaccharide component of the extracellular matrix. HA has essential functions in tissue architecture and the regulation of cell behaviour. HA turnover needs to be finely balanced. Increased HA degradation is associated with cancer, inflammation, and other pathological situations. Transmembrane protein 2 (TMEM2) is a cell surface protein that has been reported to degrade HA into ~5 kDa fragments and play an essential role in systemic HA turnover. Methods: We produced the soluble TMEM2 ectodomain (residues 106-1383; sTMEM2) in human embryonic kidney cells (HEK293) and determined its structure using X-ray crystallography. We tested sTMEM2 hyaluronidase activity using fluorescently labelled HA and size fractionation of reaction products. We tested HA binding in solution and using a glycan microarray. Results: Our crystal structure of sTMEM2 confirms a remarkably accurate prediction by AlphaFold. sTMEM2 contains a parallel ß-helix typical of other polysaccharide-degrading enzymes, but an active site cannot be assigned with confidence. A lectin-like domain is inserted into the ß-helix and predicted to be functional in carbohydrate binding. A second lectin-like domain at the C-terminus is unlikely to bind carbohydrates. We did not observe HA binding in two assay formats, suggesting a modest affinity at best. Unexpectedly, we were unable to observe any HA degradation by sTMEM2. Our negative results set an upper limit for k cat of approximately 10 -5 min -1. Conclusions: Although sTMEM2 contains domain types consistent with its suggested role in TMEM2 degradation, its hyaluronidase activity was undetectable. HA degradation by TMEM2 may require additional proteins and/or localisation at the cell surface.

Organization of the laminin polymer node.

Laminin polymerization is a key step of basement membrane assembly that depends on the binding of α, ß and γ N-terminal LN domains to form a polymer node. Nodal assembly can be divided into two steps consisting of ß- and γ-LN dimerization followed by calcium-dependent addition of the α-LN domain. The assembly and structural organization of laminin-111 LN-LEa segments was examined by size-exclusion chromatography (SEC) and electron microscopy. Triskelion-like structures were observed in negatively-stained images of purified α1/ß1/γ1 LN-LEa trimers. Image averaging of these revealed a heel-to-toe organization of the LN domains with angled outward projections of the LEa stem-like domains. A series of single-amino acid substitutions was introduced into the polymerization faces of the α1, ß1 and γ1 LN domains followed by SEC analysis to distinguish between loss of ß-γ mediated dimerization and loss of α-dependent trimerization (with intact ß-γ dimers). Dimer-blocking mutations were confined to the γ1-toe and the ß1-heel, whereas the trimer-only-blocking mutations mapped to the γ1-heel, ß1-toe and the α1-toe and heel. Thus, in the polymer node the γ1-toe pairs with the ß1-heel, the ß1-toe pairs with the α1-heel, and the α1-toe pairs with the γ1-heel.

Exploration of human xylosyltransferase for chemoenzymatic synthesis of proteoglycan linkage region.

Proteoglycans (PGs) play important roles in many biological processes including tumor progression, cell adhesion, and regulation of growth factor activities. With glycosaminoglycan chains attached to the core proteins in nature, PGs are highly challenging synthetic targets due to the difficulties in integrating the sulfated glycans with the peptide backbone. To expedite the synthesis, herein, the utility of human xylosyltransferase I (XT-I), the enzyme responsible for initiating PG synthesis, has been explored. XT-I was found to be capable of efficiently installing the xylose unit onto a variety of peptide structures on mg scales. Furthermore, an unnatural sugar, i.e., 6-azidoglucose can be transferred by XT-I introducing a reactive handle onto the glycopeptide for selective functionalization. XT-I can be coupled with ß-4-galactosyl transferase-7 for one pot synthesis of glycopeptides bearing galactose-xylose disaccharide, paving the way toward efficient chemoenzymatic synthesis of PG glycopeptides and glycoproteins.

Two-step release of kinase autoinhibition in discoidin domain receptor 1.

Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase with important functions in organogenesis and tissue homeostasis. Aberrant DDR1 activity contributes to the progression of human diseases, including fibrosis and cancer. How DDR1 activity is regulated is poorly understood. We investigated the function of the long intracellular juxtamembrane (JM) region of human DDR1 and found that the kinase-proximal segment, JM4, is an important regulator of kinase activity. Crystal structure analysis revealed that JM4 forms a hairpin that penetrates the kinase active site, reinforcing autoinhibition by the activation loop. Using in vitro enzymology with soluble kinase constructs, we established that release from autoinhibition occurs in two distinct steps: rapid autophosphorylation of the JM4 tyrosines, Tyr569 and Tyr586, followed by slower autophosphorylation of activation loop tyrosines. Mutation of JM4 tyrosines abolished collagen-induced DDR1 activation in cells. The insights may be used to develop allosteric, DDR1-specific, kinase inhibitors.

Structural biology of laminins.

Laminins are large cell-adhesive glycoproteins that are required for the formation and function of basement membranes in all animals. Structural studies by electron microscopy in the early 1980s revealed a cross-shaped molecule, which subsequently was shown to consist of three distinct polypeptide chains. Crystallographic studies since the mid-1990s have added atomic detail to all parts of the laminin heterotrimer. The three short arms of the cross are made up of continuous arrays of disulphide-rich domains. The globular domains at the tips of the short arms mediate laminin polymerization; the surface regions involved in this process have been identified by structure-based mutagenesis. The long arm of the cross is an α-helical coiled coil of all three chains, terminating in a cell-adhesive globular region. The molecular basis of cell adhesion to laminins has been revealed by recent structures of heterotrimeric integrin-binding fragments and of a laminin fragment bound to the carbohydrate modification of dystroglycan. The structural characterization of the laminin molecule is essentially complete, but we still have to find ways of imaging native laminin polymers at molecular resolution.

Laminin G-like domains: dystroglycan-specific lectins.

A unique O-mannose-linked glycan on the transmembrane protein dystroglycan binds a number of extracellular matrix proteins containing laminin G-like (LG) domains. The dystroglycan-matrix interaction is essential for muscle function: disrupted biosynthesis of the matrix-binding modification causes several forms of muscular dystrophy. The complete chemical structure of this modification has been deciphered in the past few years. We now know that LG domains bind to a glycosaminoglycan-like polysaccharide of [-3GlcAß1,3Xylα1-] units, termed matriglycan, that is attached to a highly unusual heptasaccharide linker. X-ray crystallography has revealed the principles of Ca2+-dependent matriglycan binding by LG domains. In this review, the new structural insights are applied to the growing number of LG domain-containing proteins that bind dystroglycan. It is proposed that LG domains be recognised as 'D-type' lectins to indicate their conserved function in dystroglycan binding.

Structural Basis for the Acceleration of Procollagen Processing by Procollagen C-Proteinase Enhancer-1.

Procollagen C-proteinase enhancer-1 (PCPE-1) is a secreted protein that specifically accelerates proteolytic release of the C-propeptides from fibrillar procollagens, a crucial step in fibril assembly. As such, it is a potential therapeutic target to improve tissue repair and prevent fibrosis, a major cause of mortality worldwide. Here we present the crystal structure of the active CUB1CUB2 fragment of PCPE-1 bound to the C-propeptide trimer of procollagen III (CPIII). This shows that the two CUB domains bind to two different chains of CPIII and that the N-terminal region of one CPIII chain, close to the proteolytic cleavage site, lies in the cleft between CUB1 and CUB2. This suggests that enhancing activity involves unraveling of this chain from the rest of the trimer, thus facilitating the action of the proteinase involved. Support for this hypothesis comes from site-directed mutagenesis, enzyme assays, binding studies, and molecular modeling.

Structural Basis for the Initiation of Glycosaminoglycan Biosynthesis by Human Xylosyltransferase 1.

Proteoglycans (PGs) are essential components of the animal extracellular matrix and are required for cell adhesion, migration, signaling, and immune function. PGs are composed of a core protein and long glycosaminoglycan (GAG) chains, which often specify PG function. GAG biosynthesis is initiated by peptide O-xylosyltransferases, which transfer xylose onto selected serine residues in the core proteins. We have determined crystal structures of human xylosyltransferase 1 (XT1) in complex with the sugar donor, UDP-xylose, and various acceptor peptides. The structures reveal unique active-site features that, in conjunction with functional experiments, explain the substrate specificity of XT1. A constriction within the peptide binding cleft requires the acceptor serine to be followed by glycine or alanine. The remainder of the cleft can accommodate a wide variety of sequences, but with a general preference for acidic residues. These findings provide a framework for understanding the selectivity of GAG attachment.

Structural and functional analysis of two small leucine-rich repeat proteoglycans, fibromodulin and chondroadherin.

The small leucine-rich proteoglycans (SLRPs) are important regulators of extracellular matrix assembly and cell signalling. We have determined crystal structures at ~2.2Å resolution of human fibromodulin and chondroadherin, two collagen-binding SLRPs. Their overall fold is similar to that of the prototypical SLRP, decorin, but unlike decorin neither fibromodulin nor chondroadherin forms a stable dimer. A previously identified binding site for integrin α2ß1 maps to an α-helix in the C-terminal cap region of chondroadherin. Interrogation of the Collagen Toolkits revealed a unique binding site for chondroadherin in collagen II, and no binding to collagen III. A triple-helical peptide containing the sequence GAOGPSGFQGLOGPOGPO (O is hydroxyproline) forms a stable complex with chondroadherin in solution. In fibrillar collagen I and II, this sequence is aligned with the collagen cross-linking site KGHR, suggesting a role for chondroadherin in cross-linking.

Crystal Structure of the Heterotrimeric Integrin-Binding Region of Laminin-111.

Laminins are cell-adhesive glycoproteins that are essential for basement membrane assembly and function. Integrins are important laminin receptors, but their binding site on the heterotrimeric laminins is poorly defined structurally. We report the crystal structure at 2.13 Å resolution of a minimal integrin-binding fragment of mouse laminin-111, consisting of ∼50 residues of α1ß1γ1 coiled coil and the first three laminin G-like (LG) domains of the α1 chain. The LG domains adopt a triangular arrangement, with the C terminus of the coiled coil situated between LG1 and LG2. The critical integrin-binding glutamic acid residue in the γ1 chain tail is surface exposed and predicted to bind to the metal ion-dependent adhesion site in the integrin ß1 subunit. Additional contacts to the integrin are likely to be made by the LG1 and LG2 surfaces adjacent to the γ1 chain tail, which are notably conserved and free of obstructing glycans.

Crystallographic analysis of the laminin ß2 short arm reveals how the LF domain is inserted into a regular array of LE domains.

Laminins are a major constituent of all basement membranes. The polymerisation of laminins at the cell surface is mediated by the three short arms of the cross-shaped laminin heterotrimer. The short arms contain repeats of laminin-type epidermal growth factor-like (LE) domains, interspersed with globular domains of unknown function. A single LF domain is inserted between LE5 and LE6 of the laminin ß1 and ß2 chains. We report the crystal structure at 1.85Å resolution of the laminin ß2 LE5-LF-LE6 region. The LF domain consists of a ß-sandwich related to bacterial family 35 carbohydrate binding modules, and more distantly to the L4 domains present in the short arms of laminin α and γ chains. An α-helical region mediates the extensive interaction of the LF domain with LE5. The relative arrangement of LE5 and LE6 is very similar to that of consecutive LE domains in uninterrupted LE tandems. Fitting atomic models to a low-resolution structure of the first eight domains of the laminin ß1 chain determined by small-angle X-ray scattering suggests a deviation from the regular LE array at the LE4-LE5 junction. These results advance our understanding of laminin structure.

Panels of chemically-modified heparin polysaccharides and natural heparan sulfate saccharides both exhibit differences in binding to Slit and Robo, as well as variation between protein binding and cellular activity.

Heparin/heparan sulfate (HS) glycosaminoglycans are required for Slit-Robo cellular responses. Evidence exists for interactions between each combination of Slit, Robo and heparin/HS and for formation of a ternary complex. Heparin/HS are complex mixtures displaying extensive structural diversity. The relevance of this diversity has been studied to a limited extent using a few select chemically-modified heparins as models of HS diversity. Here we extend these studies by parallel screening of structurally diverse panels of eight chemically-modified heparin polysaccharides and numerous natural HS oligosaccharide chromatographic fractions for binding to both Drosophila Slit and Robo N-terminal domains and for activation of a chick retina axon response to the Slit fragment. Both the polysaccharides and oligosaccharide fractions displayed variability in binding and cellular activity that could not be attributed solely to increasing sulfation, extending evidence for the importance of structural diversity to natural HS as well as model modified heparins. They also displayed differences in their interactions with Slit compared to Robo, with Robo preferring compounds with higher sulfation. Furthermore, the patterns of cellular activity across compounds were different to those for binding to each protein, suggesting that biological outcomes are selectively determined in a subtle manner that does not simply reflect the sum of the separate interactions of heparin/HS with Slit and Robo.

Structural basis of laminin binding to the LARGE glycans on dystroglycan.

Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in skeletal muscle and the nervous system. Reduced matrix binding by α-dystroglycan (α-DG) due to perturbed glycosylation is a pathological feature of several forms of muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) synthesizes the matrix-binding heteropolysaccharide [-glucuronic acid-ß1,3-xylose-α1,3-]n. Using a dual exoglycosidase digestion, we confirm that this polysaccharide is present on native α-DG from skeletal muscle. The atomic details of matrix binding were revealed by a high-resolution crystal structure of laminin-G-like (LG) domains 4 and 5 (LG4 and LG5) of laminin-α2 bound to a LARGE-synthesized oligosaccharide. A single glucuronic acid-ß1,3-xylose disaccharide repeat straddles a Ca(2+) ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca(2+)-bound water molecules. The chelating binding mode accounts for the high affinity of this protein-carbohydrate interaction. These results reveal a previously uncharacterized mechanism of carbohydrate recognition and provide a structural framework for elucidating the mechanisms underlying muscular dystrophy.

Insights into Collagen Uptake by C-type Mannose Receptors from the Crystal Structure of Endo180 Domains 1-4.

The C-type mannose receptor and its homolog Endo180 (or uPARAP, for urokinase plasminogen activator receptor-associated protein) mediate the endocytic uptake of collagen by macrophages and fibroblasts. This process is required for normal tissue remodeling, but also facilitates the growth and dissemination of tumors. We have determined the crystal structure at 2.5 Å resolution of the N-terminal region of Endo180, consisting of a ricin-like domain, a fibronectin type II (FN2) domain, and two C-type lectin (CTL) domains. The L-shaped arrangement of these domains creates a shallow trench spanning the FN2 and CTL1 domains, which was shown by mutagenesis to bind triple-helical and denatured collagen. Small-angle X-ray scattering showed that the L-shaped structure is maintained in solution at neutral and acidic pH, irrespective of calcium ion loading. Collagen binding was equally unaffected by acidic pH, suggesting that collagen release in endosomes is not regulated by changes within the Endo180 N-terminal region.

Signaling switch of the axon guidance receptor Robo3 during vertebrate evolution.

Development of neuronal circuits is controlled by evolutionarily conserved axon guidance molecules, including Slits, the repulsive ligands for roundabout (Robo) receptors, and Netrin-1, which mediates attraction through the DCC receptor. We discovered that the Robo3 receptor fundamentally changed its mechanism of action during mammalian evolution. Unlike other Robo receptors, mammalian Robo3 is not a high-affinity receptor for Slits because of specific substitutions in the first immunoglobulin domain. Instead, Netrin-1 selectively triggers phosphorylation of mammalian Robo3 via Src kinases. Robo3 does not bind Netrin-1 directly but interacts with DCC. Netrin-1 fails to attract pontine neurons lacking Robo3, and attraction can be restored in Robo3(-/-) mice by expression of mammalian, but not nonmammalian, Robo3. We propose that Robo3 evolution was key to sculpting the mammalian brain by converting a receptor for Slit repulsion into one that both silences Slit repulsion and potentiates Netrin attraction.

Signalling complexes at the cell-matrix interface.

The extracellular matrix critically controls cell behaviour. Many cell-matrix interactions are mediated by transmembrane receptors of the integrin family. In the last two years, the structural changes resulting from ligand binding to integrins α5ß1, αvß3 and αIIbß3 have been mapped in unprecedented detail. The structure of integrin αXß2 has revealed how ligand binding to the α I domain is transmitted to the rest of the ectodomain. The structural characterisation of the cytosolic regulator talin has been continued, revealing how the integrin binding site is blocked in auto-inhibited talin. Finally, structures of the discoidin domain receptors DDR1 and DDR2 have begun to reveal how these atypical receptor tyrosine kinases become activated by the major matrix component collagen.

Biologically-active laminin-111 fragment that modulates the epithelial-to-mesenchymal transition in embryonic stem cells.

The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the ß1-chain-derived fragment interacts via α3ß1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs--key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the α3ß1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9.

Normal activation of discoidin domain receptor 1 mutants with disulfide cross-links, insertions, or deletions in the extracellular juxtamembrane region: mechanistic implications.

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by collagen. DDR activation does not appear to occur by the common mechanism of ligand-induced receptor dimerization: the DDRs form stable noncovalent dimers in the absence of ligand, and ligand-induced autophosphorylation of cytoplasmic tyrosines is unusually slow and sustained. Here we sought to identify functionally important dimer contacts within the extracellular region of DDR1 by using cysteine-scanning mutagenesis. Cysteine substitutions close to the transmembrane domain resulted in receptors that formed covalent dimers with high efficiency, both in the absence and presence of collagen. Enforced covalent dimerization did not result in constitutive activation and did not affect the ability of collagen to induce receptor autophosphorylation. Cysteines farther away from the transmembrane domain were also cross-linked with high efficiency, but some of these mutants could no longer be activated. Furthermore, the extracellular juxtamembrane region of DDR1 tolerated large deletions as well as insertions of flexible segments, with no adverse effect on activation. These findings indicate that the extracellular juxtamembrane region of DDR1 is exceptionally flexible and does not constrain the basal or ligand-activated state of the receptor. DDR1 transmembrane signaling thus appears to occur without conformational coupling through the juxtamembrane region, but requires specific receptor interactions farther away from the cell membrane. A plausible mechanism to explain these findings is signaling by DDR1 clusters.

Heterotropic modulation of selectin affinity by allosteric antibodies affects leukocyte rolling.

Selectins are a family of adhesion receptors designed for efficient leukocyte tethering to the endothelium under shear. As a key property to resist premature bond disruption, selectin adhesiveness is enhanced by tensile forces that promote the conversion of a bent into an extended conformation of the N-terminal lectin and epidermal growth factor-like domains. Conformation-specific Abs have been invaluable in deciphering the activation mechanism of integrins, but similar reagents are not available for selectins. In this study, we show that the anti-human L-selectin mAbs DREG-55 and LAM1-5 but not DREG-56, DREG-200, or LAM1-1 heterotropically modulate adhesion presumably by stabilizing the extended receptor conformation. Force-free affinity assays, flow chamber, and microkinetic studies reveal a ligand-specific modulation of L-selectin affinity by DREG-55 mAb, resulting in a dramatic decrease of rolling velocity under flow. Furthermore, secondary tethering of polymorphonuclear cells was blocked by DREG-200 but significantly boosted by DREG-55 mAb. The results emphasize the need for a new classification for selectin Abs and introduce the new concept of heterotropic modulation of receptor function.