Isolation of a native osteoblast matrix with a specific affinity for BMP2

During their commitment and differentiation toward the osteoblast lineage, mesenchymal stem cells secrete a unique extracellular matrix (ECM) that contains large quantities of glycosaminoglycans (GAGs). Proteoglycans (PGs) are major structural and functional components of the ECM and are composed of a core protein to which one or more glycosaminoglycan sugar chains (GAGs) attach. The association of BMP2, a member of the TGF-β super-family of growth factors, and a known heparin-binding protein, with GAGs has been implicated as playing a significant role in modulating the growth factor’s in vitro bioactivity. Here we have characterised an osteoblast-derived matrix (MX) obtained from decellularised MC3T3-E1 cell monolayers for its structural attributes, using SEM and histology, and for its functional ability to maintain cell growth and viability. Using a combination of histology and anion exchange chromatography, we first confirmed the retention of GAGs within MX following the decellularisation process. Then the binding specificity of the retained GAG species within the MX for BMP2 was examined using a BMP2-HBP/EGFP (BMP2 Heparin-Binding Peptide/Enhanced Green Fluorescent Protein) fusion protein. The results of this study provide further evidence for a central role of the ECM in the regulation of BMP2 bioactivity, hence on mesenchymal stem cell commitment to the osteoblast lineage.     

Calreticulin, PDI, Grp94 and BiP chaperone proteins are associated with retained COMP in pseudoachondroplasia chondrocytes.

Cartilage oligomeric matrix protein (COMP), a large pentameric glycoprotein and member of the thrombospondin (TSP) group of extracellular proteins, is found in the territorial matrix surrounding chondrocytes. More than 50 unique COMP mutations have been identified as causing two skeletal dysplasias: pseudoachondroplasia (PSACH); and multiple epiphyseal dysplasia (EDM1). Recent studies suggest that calcium-binding and calcium-induced protein folding differ between wild type and mutant proteins, and abnormal processing of the mutant COMP protein contributes to the characteristic enlarged lamellar appearing rER cisternae in PSACH and EDMI chondrocytes in vivo and in vitro. Towards the goal of delineating the pathogenesis of PSACH and EDM1, in-vivo PSACH growth plate and in-vitro PSACH chondrocytes cultured in alginate beads were examined to identify and localize the chaperone proteins participating in the processing of the retained extracellular matrix proteins in the PSACH rER. Aggrecan was localized to both the rER cisternae and matrix while COMP and type IX collagen were only found in the rER. Type II collagen was solely found in the ECM suggesting that it is processed and transported differently from other retained ECM proteins. Five chaperone proteins: BiP (Grp78); calreticulin (CRT); protein disulfide (PDI); ERp72; and Grp94, demonstrated immunoreactivity in the enlarged PSACH cisternae and the short rER channels of chondrocytes from both in-vivo and in-vitro samples. The chaperone proteins cluster around the electron dense material within the enlarged rER cisternae. CRT, PDI and GRP94 AB-gold particles appear to be closely associated with COMP. Immunoprecipitation and Western blot, and Fluorescence Resonance Energy Transfer (FRET) analyses indicate that CRT, PDI and GRP94 are in close proximity to normal and mutant COMP and BiP to mutant COMP. These results suggest that these proteins play a role in the processing and transport of wild type COMP in normal chondrocytes and in the retention of mutant COMP in PSACH chondrocytes.     

Galectin 1 inhibits incorporation of vitronectin and chondroitin sulfate B into the extracellular matrix of human vascular smooth muscle cells

Galectin-1, a beta-galactoside-binding dimeric lectin, interacts with the extracellular matrix (ECM) of smooth muscle cells (SMCs) and with particular ECM proteins. Enrichment of the ECM with galectin-1 affects adhesion and proliferation of cultured SMCs. Here we investigated whether galectin-1 (1) interacts with glycosaminoglycan (GAG) chains, (2) cross-links between ligands and facilitates the incorporation of GAGs, vitronectin and plasma fibronectin in the ECM of vascular SMCs. A recombinant galectin-1 fusion protein GalH, used in this study, formed dimers and interacted with ECM proteins. GAG chains inhibited these interactions. Among the studied GAG chains, only chondroitin sulfate B interacted with GalH in beta-galactoside-dependent manner. GalH did not bridge between ECM proteins on solid phase and [125I]-labelled ECM proteins or GAGs in solution. The ECM incorporated less vitronectin in the presence of soluble GalH. GalH-enriched ECM incorporated less vitronectin and chondroitin sulfate B. The ECM partially depleted of endogenous galectins incorporated more chondroitin sulfate B compared to untreated ECM. These results suggest that galectin-1 is likely to be involved in the ECM assembly affecting incorporation of some ECM components important for SMC behaviour.     

Characterization of glycosaminoglycans during tooth development and mineralization in the axolotl, Ambystoma mexicanum

Glycosaminoglycans (GAGs) involved in the formation of the teeth of Ambystoma mexicanum were located and characterized with the cuprolinic blue (CB) staining method and transmission electron microscopy (TEM). Glycosaminoglycan-cuprolinic blue precipitates (GAGCB) were found in different compartments of the mineralizing tissue. Various populations of elongated GAGCB could be discriminated both according to their size and their preferential distribution in the extracellular matrix (ECM). GAGCB populations that differ in their composition could be attributed not only to the compartments of the ECM but also to different zones and to different tooth types (early-larval and transformed). Larger precipitates were only observed within the dentine matrix of the shaft of the early-larval tooth. The composition of the populations differed significantly between the regions of the transformed tooth: pedicel, shaft and dividing zone. In later stages of tooth formation, small-sized GAGCBs were seen as intracellular deposits in the ameloblasts. It is concluded that the composition of GAGCB populations seems to play a role in the mineralization processes during tooth development in A. mexicanum and influence qualitative characteristics of the mineral in different tooth types and zones, and it is suggested that GAGs might be resorbed by the enamel epithelium during the late phase of enamel formation.     

Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase

Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.     

Selective intracellular retention of extracellular matrix proteins and chaperones associated with pseudoachondroplasia.

Mutations in the cartilage oligomeric matrix protein (COMP) gene result in pseudoachondroplasia (PSACH), which is a chondrodysplasia characterized by early-onset osteoarthritis and short stature. COMP is a secreted pentameric glycoprotein that belongs to the thrombospondin family of proteins. We have identified a novel missense mutation which substitutes a glycine for an aspartic acid residue in the thrombospondin (TSP) type 3 calcium-binding domain of COMP in a patient diagnosed with PSACH. Immunohistochemistry and immunoelectron microscopy both show abnormal retention of COMP within characteristically enlarged rER inclusions of PSACH chondrocytes, as well as retention of fibromodulin, decorin and types IX, XI and XII collagen. Aggrecan and types II and VI collagen were not retained intracellularly within the same cells. In addition to selective extracellular matrix components, the chaperones HSP47, protein disulfide isomerase (PDI) and calnexin were localized at elevated levels within the rER vesicles of PSACH chondrocytes, suggesting that they may play a role in the cellular retention of mutant COMP molecules. Whether the aberrant rER inclusions in PSACH chondrocytes are a direct consequence of chaperone-mediated retention of mutant COMP or are otherwise due to selective intracellular protein interactions, which may in turn lead to aggregation within the rER, is unclear. However, our data demonstrate that retention of mutant COMP molecules results in the selective retention of ECM molecules and molecular chaperones, indicating the existence of distinct secretory pathways or ER-sorting mechanisms for matrix molecules, a process mediated by their association with various molecular chaperones.     

Interactions between M proteins of Streptococcus pyogenes and glycosaminoglycans promote bacterial adhesion to host cells.

Several microbial pathogens have been reported to interact with glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix. Here we demonstrate that M protein, a major surface-expressed virulence factor of the human bacterial pathogen, Streptococcus pyogenes, mediates binding to various forms of GAGs. Hence, S. pyogenes strains expressing a large number of different types of M proteins bound to dermatan sulfate (DS), highly sulfated fractions of heparan sulfate (HS) and heparin, whereas strains deficient in M protein surface expression failed to interact with these GAGs. Soluble M protein bound DS directly and could also inhibit the interaction between DS and S. pyogenes. Experiments with M protein fragments and with streptococci expressing deletion constructs of M protein, showed that determinants located in the NH2-terminal part as well as in the C-repeat region of the streptococcal proteins are required for full binding to GAGs. Treatment with ABC-chondroitinase and HS lyase that specifically remove DS and HS chains from cell surfaces, resulted in significantly reduced adhesion of S. pyogenes bacteria to human epithelial cells and skin fibroblasts. Together with the finding that exogenous DS and HS could inhibit streptococcal adhesion, these data suggest that GAGs function as receptors in M protein-mediated adhesion of S. pyogenes.     

Regulation of glycosaminoglycan biogenesis is critical for sensitive‐period‐dependent vocal ontogeny

In the brain, the extracellular matrix (ECM) plays a central role during neural development and thus modulates critical‐period regulated behavioral ontogeny. The major components of the ECM are glycosaminoglycans (GAGs) including chondroitin sulfate (CS). However, the specific roles of GAGs in behavioral development are largely unknown. It has been shown that xylosides affect the biological functions of GAGs through modulating GAG biosynthesis. Particularly, xylosides affect GAG biosynthesis through priming of GAG chains (priming activity), competing with endogenous core proteins that carry GAG initiation sites (decoy activity), or both. Using birdsong as our model, we investigated, for the first time, how xyloside‐mediated modulation of GAG biogenesis affects song development. Xylosides infused into motor cortex of juvenile birds alter song development by specifically affecting ontogeny of the stereotyped sequence rather than the acoustic structure of syllables. Further analyses reveal that observed changes can be attributed to the priming activity rather than the decoy activity of xylosides. Collectively, these results suggest that regulation of GAG biogenesis through chemical biology approaches may allow promising therapeutic interventions of critical‐period‐dependent central nervous system plasticity. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1401–1412, 2017     

Anchorage of collagen-tailed acetylcholinesterase to the extracellular matrix is mediated by heparan sulfate proteoglycans

Heparan sulfate and heparin, two sulfated glycosaminoglycans (GAGs), extracted collagen-tailed acetylcholinesterase (AChE) from the extracellular matrix (ECM) of the electric organ of Discopyge tschudii. The effect of heparan sulfate and heparin was abolished by protamine; other GAGs could not extract the esterase. The solubilization of the asymmetric AChE apparently occurs through the formation of a soluble AChE-GAG complex of 30S. Heparitinase treatment but not chondroitinase ABC treatment of the ECM released asymmetric AChE forms. This provides direct evidence for the vivo interaction between asymmetric AChE and heparan sulfate residues of the ECM. Biochemical analysis of the electric organ ECM showed that sulfated GAGs bound to proteoglycans account for 5% of the total basal lamina. Approximately 20% of the total GAGs were susceptible to heparitinase or nitrous acid oxidation which degrades specifically heparan sulfates, and approximately 80% were susceptible to digestion with chondroitinase ABC, which degrades chondroitin-4 and -6 sulfates and dermatan sulfate. Our experiments provide evidence that asymmetric AChE and carbohydrate components of proteoglycans are associated in the ECM; they also indicate that a heparan sulfate proteoglycan is involved in the anchorage of the collagen-tailed AChE to the synaptic basal lamina.     

Sorsby's fundus dystrophy tissue inhibitor of metalloproteinases-3 (TIMP-3) mutants have unimpaired matrix metalloproteinase inhibitory activities, but affect cell adhesion to the extracellular matrix.

The TIMP family of matrix metalloproteinase inhibitors consists of four members, of which TIMP-1, -2 and -4 are secreted, freely diffusible proteins, whereas TIMP-3 is ECM-associated. Mutations in the TIMP3 gene have been linked to Sorsby's fundus dystrophy (SFD), an autosomal dominant inherited retinal degenerative disease that leads to blindness. The SFD mutations characterized result in introduction of an unpaired cysteine residue in the C-terminal domain of TIMP-3. We have expressed four SFD mutant TIMP-3 proteins in baby hamster kidney (BHK) cells and evaluated their characteristics alongside wild-type TIMP-3. Analysis of the mutant proteins (Ser156Cys, Gly167Cys, Tyr168Cys and Ser181Cys) by SDS-PAGE and reverse zymography revealed that each of the mutants retained gelatinase A and gelatinase B inhibitory activity, and were localized to the ECM. Association rate constants for Ser156Cys TIMP-3 with gelatinase-A, gelatinase-B, stromelysin-1 and collagenase-3 were only moderately reduced compared to wild-type TIMP-3. However, all of the mutants displayed aberrant protein-protein interactions, resulting in the presence of additional proteins or complexes in ECM preparations. Two of the mutants (Ser156Cys and Ser181Cys) showed a marked propensity to form multiple higher molecular-weight complexes that retained TIMP activity on reverse zymography. Expression of the SFD mutant TIMP-3 (and to a lesser extent, wild-type TIMP-3) proteins in BHK cells conferred increased cell adhesiveness to the ECM. Our findings indicate that the pathogenesis of Sorsby's fundus dystrophy cannot be attributed to a failure to localize SFD TIMP-3 proteins to the ECM or defects in MMP inhibition, but may involve the formation of aberrant TIMP-3-containing protein complexes and altered cell adhesion.     

Impact of Mycobacterium ulcerans biofilm on transmissibility to ecological niches and Buruli ulcer pathogenesis

The role of biofilms in the pathogenesis of mycobacterial diseases remains largely unknown. Mycobacterium ulcerans, the etiological agent of Buruli ulcer, a disfiguring disease in humans, adopts a biofilm-like structure in vitro and in vivo, displaying an abundant extracellular matrix (ECM) that harbors vesicles. The composition and structure of the ECM differs from that of the classical matrix found in other bacterial biofilms. More than 80 proteins are present within this extracellular compartment and appear to be involved in stress responses, respiration, and intermediary metabolism. In addition to a large amount of carbohydrates and lipids, ECM is the reservoir of the polyketide toxin mycolactone, the sole virulence factor of M. ulcerans identified to date, and purified vesicles extracted from ECM are highly cytotoxic. ECM confers to the mycobacterium increased resistance to antimicrobial agents, and enhances colonization of insect vectors and mammalian hosts. The results of this study support a model whereby biofilm changes confer selective advantages to M. ulcerans in colonizing various ecological niches successfully, with repercussions for Buruli ulcer pathogenesis.     

Glycosaminoglycan composition changes with MG-63 osteosarcoma osteogenesis in vitro and induces human mesenchymal stem cell aggregation

Osteogenic differentiation is coordinated by the exposure of cells to temporal changes in a combination of growth factors and elements within the extracellular matrix (ECM). Many of the key proteins that drive these changes share the property of being dependent on ECM glycosaminoglycans (GAGs) for their activity. Here, we examined whether GAGs isolated from proliferating, differentiating and mineralizing MG-63 osteosarcoma cells differed in their physical properties, and thus in their capacities to coordinate the osteogenic cascade both in human MG-63 osteosarcoma cells and primary human mesenchymal stem cells (hMSCs). Our results show that the size distribution of GAGs, the expression of GAG-carrying proteoglycan cores and the expression of enzymes involved in their modification systematically change as MG-63 cells mature in culture. When dosed back onto cells exogenously in soluble form, GAGs regulated MG-63 survival and growth in a dose-dependent manner, but not differentiation in either cell type. In contrast, hMSCs aggregated into distinct colonies when grown on GAG-coated substrates, while MG-63 cells did not. Heparin-coated substrates improved hMSC viability without inducing aggregation. These results suggest a complex role for GAGs in coordinating the emergence of the osteoblast phenotype, and provide further evidence for the use of heparans in bone tissue repair applications.     

Expression of PSACH-associated mutant COMP in tendon fibroblasts leads to increased apoptotic cell death irrespective of the secretory characteristics of mutant COMP.

OBJECTIVE: Pseudoachondroplasia (PSACH) is a dominantly inherited chondrodysplasia associated with mutations of cartilage oligomeric matrix protein (COMP), characterized clinically by disproportionate dwarfism and laxity of joints and ligaments. Studies in chondrocytes and cartilage biopsies suggest that the cartilage disease is caused by retention of mutant COMP in the endoplasmic reticulum of chondrocytes and by disruption of the collagen network of the extracellular matrix. The pathogenesis of the tendon disease remains unclear in the absence of a cell culture model, with available tendon biopsies leading to conflicting results with respect to the intracellular retention of mutant COMP. METHODS: We established a cell culture model using adenoviral gene transfer in tendon fibroblast cultures. We compared the effect of expression of three PSACH-associated COMP mutants and the wildtype protein on COMP secretion, matrix composition and cellular viability. RESULTS: Our results show that mutants D475N and D469Delta are retained within the endoplasmic reticulum of tendon cells similar to what is known from chondrocytes, whereas mutant H587R is secreted like wildtype COMP. In spite of this difference, the collagen I matrix formed in culture appears disturbed for all three mutants. All COMP-mutants induce apoptotic cell death irrespective of their differing secretion patterns. CONCLUSION: Pathogenic pathways leading to tendon disease in humans appear to be heterogeneous between different COMP mutants.     

Vaccinia virus 4c (A26L) protein on intracellular mature virus binds to the extracellular cellular matrix laminin

Vaccinia virus intracellular mature virus (IMV) binds to glycosaminoglycans (GAGs) on cells via three virion proteins, H3L, A27L, and D8L. In this study, we demonstrated that binding of IMV to BSC40 cells was competitively inhibited by soluble laminin but not by fibronectin or collagen V, suggesting that this cell surface extracellular matrix (ECM) protein may play a role in vaccinia virus entry. Moreover, IMV infection of GAG(-) sog9 cells was also inhibited by laminin, demonstrating that virion binding to laminin does not involve a prior interaction with GAGs. Furthermore, comparative envelope protein analyses of wild-type vaccinia virus strain Western Reserve, which binds to laminin, and of a mutant virus, IA27L, which does not, showed that the A26L open reading frame (ORF), encoding an envelope protein, was mutated in IA27L, resulting in A26L being absent from the IMV. Expression of the wild-type A26L ORF in IA27L resulted in laminin binding activity. Moreover, recombinant A26L protein bound to laminin in vitro with a high affinity, providing direct evidence that A26L is the laminin binding protein on IMV. In summary, these results reveal a novel role for the vaccinia viral envelope protein A26L in binding to the ECM protein laminin, an association that is proposed to facilitate IMV entry.     

Enzymatic Degradation of GlycosaminogIycans

Abstract

Glycosarninoglycans (GAGs) play an intricate role in the extracellular matrix (ECM), not only as soluble components and polyelectrolytes, but also by specific interactions with growth factors and other transient components of the ECM. Modifications of GAG chains, such as isomerization, sulfation, and acetylation, generate the chemical specificity of GAGs. GAGS can be depolymerized enzymatically either by eliminative cleavage with lyases (EC 4.2.2.-) or by hydrolytic cleavage with hydrolases (EC 3.2.1.-). Often, these enzymes are specific for residues in the polysaccharide chain with certain modifications. As such, the enzymes can serve as tools for studying the physiological effect of residue modifications and as models at the molecular level of protein-GAG recognition. This review examines the structure of the substrates, the properties of enzymatic degradation, and the enzyme substrate-interactions at a molecular level. The primary structure of several GAGS is organized macro-scopicallyby segregation into alternating blocks of specific sulfation patterns and microscopicallyby formation of oligosaccharide sequences with specific binding functions. Among GAGs, considerable dermatan sulfate, heparin and heparan sulfate show conformational flexibility in solution. They elicit sequence-specific interactions with enzymes that degrade them, as well as with other proteins, however, the effect of conformational flexibility on protein-GAG interactions is not clear. Recent findings have established empirical rules of substrate specificity and elucidated molecular mechanisms of enzyme-substrate interactions for enzymes that degrade GAGs. Here we propose that local formation of polysaccharide secondary structure is determined by the immediate sequence environment within the GAG polymer, and that this secondary structure, in turn, governs the binding and catalytic interactions between proteins and GAGs.     

Glycosaminoglycans contribute to extracellular matrix fiber recruitment and arterial wall mechanics

Elastic and collagen fibers are well known to be the major load-bearing extracellular matrix (ECM) components of the arterial wall. Studies of the structural components and mechanics of arterial ECM generally focus on elastin and collagen fibers, and glycosaminoglycans (GAGs) are often neglected. Although GAGs represent only a small component of the vessel wall ECM, they are considerably important because of their diverse functionality and their role in pathological processes. The goal of this study was to study the mechanical and structural contributions of GAGs to the arterial wall. Biaxial tensile testing was paired with multiphoton microscopic imaging of elastic and collagen fibers in order to establish the structure–function relationships of porcine thoracic aorta before and after enzymatic GAG removal. Removal of GAGs results in an earlier transition point of the nonlinear stress–strain curves \((p<0.05)\). However, stiffness was not significantly different after GAG removal treatment, indicating earlier but not absolute stiffening. Multiphoton microscopy showed that when GAGs are removed, the adventitial collagen fibers are straighter, and both elastin and collagen fibers are recruited at lower levels of strain, in agreement with the mechanical change. The amount of stress relaxation also decreased in GAG-depleted arteries \((p<0.05)\). These findings suggest that the interaction between GAGs and other ECM constituents plays an important role in the mechanics of the arterial wall, and GAGs should be considered in addition to elastic and collagen fibers when studying arterial function.     

Contributions of Glycosaminoglycans to Collagen Fiber Recruitment in Constitutive Modeling of Arterial Mechanics

The contribution of glycosaminoglycans (GAGs) to the biological and mechanical functions of biological tissue has emerged as an important area of research. GAGs provide structural basis for the organization and assembly of extracellular matrix (ECM). The mechanics of tissue with low GAG content can be indirectly affected by the interaction of GAGs with collagen fibers, which have long been known to be one of the primary contributors to soft tissue mechanics. Our earlier study showed that enzymatic GAG depletion results in straighter collagen fibers that are recruited at lower levels of stretch, and a corresponding shift in earlier arterial stiffening (Mattson et al., 2016). In this study, the effect of GAGs on collagen fiber recruitment was studied through a structure-based constitutive model. The model incorporates structural information, such as fiber orientation distribution, content, and recruitment of medial elastin, medial collagen, and adventitial collagen fibers. The model was first used to study planar biaxial tensile stress-stretch behavior of porcine descending thoracic aorta. Changes in elastin and collagen fiber orientation distribution, and collagen fiber recruitment were then incorporated into the model in order to predict the stress-stretch behavior of GAG depleted tissue. Our study shows that incorporating early collagen fiber recruitment into the model predicts the stress-stretch response of GAG depleted tissue reasonably well (rms = 0.141); considering further changes of fiber orientation distribution does not improve the predicting capability (rms = 0.149). Our study suggests an important role of GAGs in arterial mechanics that should be considered in developing constitutive models.     

Substitution of two histidine residues in YadA protein of Yersinia enterocolitica abrogates collagen binding, cell adherence and mouse virulence

The plasmid-encoded surface protein YadA of Yersinia enterocolitica mediates binding to diverse extracellular matrix (ECM) proteins, adherence to epithelial cell lines, resistance to complement lysis, autoagglutination, and is required for mouse virulence. Using site-directed mutagenesis we attempted to analyse the relationship between structural domains and functions of YadA. In a first approach we could abrogate collagen binding by chemical modification of histidyl residues of YadA protein. This result prompted us to substitute histidyl residues (His) of conserved regions of YadA protein of Y. enterocolitica 08 by tyrosine residues using site-directed mutagenesis. Substitution of His-156 and His-159 (YadA-2 mutant) resulted in abrogation of binding to ECM proteins, of cell adherence, and in reduction of mouse virulence, whereas autoagglutination, serum complement resistance and oligomer formation remained unaffected. A striking result was obtained from the orogastric mouse-infection model: the YadA-2 mutant retained the ability to colonize the small intestine and to invade and multiply within the Peyer's patches but was impaired in colonizing mesenteric lymph nodes and spleen in comparison to the wild-type strain.     

Extracellular matrix: from atomic resolution to ultrastructure

The extracellular matrix (ECM) is a highly organized multimolecular structure, essential for life in higher organisms. Although substantial high-resolution structural information is available for relatively small fragments of ECM components, the inherent difficulty in preparing and analyzing samples of large, fibrous polymers impedes structural efforts. Here, we review recent advances in understanding the structure of three important ECM components: collagen, fibrillin and fibronectin. Emphasis is placed on the key role of intermolecular interactions in assembling larger, microm scale, structures.     

Erythrocytes express chondroitin sulphate/dermatan sulphate, which undergoes quantitative changes during diabetes and mediate erythrocyte adhesion to extracellular matrix components

Glycosaminoglycans (GAGs) such as chondroitin sulphate/dermatan sulphate (CS/DS) are complex molecules that are widely expressed on the cell membrane and extracellular matrix (ECM). They play an important role in wide range of biological activities especially during pathological conditions. Diabetes, a metabolic disorder characterized by sustained hyperglycemia, is known to affect GAGs in different tissues and affect erythrocyte adhesion. The present investigation was aimed at exploring the nature of GAGs present in erythrocytes and its role on adhesion of erythrocytes from control and diabetic rats to major extracellular matrix components. GAGs isolated from erythrocytes were demonstrated to be CS/DS and a 2-fold increase was observed in erythrocytes from diabetic rats. Disaccharide composition analysis by HPLC after depolymerization by the enzyme, chondroitinase ABC showed the presence of 4-O sulphated disaccharide units with small amounts of non-sulphated disaccharides, in both control and diabetic erythrocytes. Erythrocytes from diabetic rats, however, showed significantly increased binding to poly-l-ornithine (P-orn), type IV collagen, laminin and fibronectin, which was abrogated on treatment with chondroitinase ABC to various degrees. This study sheds new light on CS/DS in erythrocytes and its likely biological implications in vivo.