Regulated expression of keratan sulphate and peanut agglutinin binding sites during organogenesis in the developing chick

 Keratan sulphate proteoglycans are potentially important during development and are possible binding molecules for the lectin, peanut agglutinin, a marker for areas that are inhibitory for axonal growth in early embryos. The present study describes the spatiotemporal distributions of keratan sulphate epitopes and peanut agglutinin binding sites during organogenesis in the developing chick from E5 to hatching. The widespread distributions of these molecules did not often overlap but clearly delimited different carbohydrate compartments demonstrating that peanut agglutinin does not necessarily bind to keratan sulphate proteoglycans. These markers were mostly extracellular but keratan sulphate, in particular, was found within certain specific cells in cartilage, gonad, heart and pancreas, at certain ages. The presence of keratan sulphate in putative germ cells during their migrations and in the gonads may be of particular importance. Their distributions generally evoke modulation of adhesion allowing cell migrations or morphogenetic movements related to epitheliomesenchymal interactions, but may also suggest an involvement in axonal guidance in skin, cartilage, gut and possibly heart. Furthermore, in the kidney, peanut agglutinin binding sites seem to be related to the functional differentiation of the nephrons. Accepted: 23 February 1998     

Form and function of arabinogalactans and arabinogalactan-proteins

The occurrence, isolation, chemistry and physico-chemistry of plant arabino-3,6-galactans and arabino-3,6-galactan-proteins is reviewed. The structural relationships between arabino-3,6-galactans from gymnosperm wood, gum exudates of Acacia and other trees, and from plant callus cells and whole tissues are discussed. The nature of these proteoglycans is compared with the arabinose and galactose containing cell wall glycoproteins. Interactions of the arabino-3,6-galactan proteoglycans with carbohydrate binding proteins and with Yariv antigens are described. The utility of these reactions for both cellular and subcellular localization of the proteoglycans is discussed. The possible biological roles of the arabinogalactans and the arabinogalactan-proteins are reviewed.     

Autoimmune response in cartilage-delivered peptides in a patient with osteoarthritis

Whatever the initiating factor of osteoarthritis (OA), the process ultimately unmasks the immunogenic determinants of chondrocytes, proteoglycans and collagens, which then triggers autoimmune reactions. Although the precise mechanism of the immune responses in the pathogenesis of OA requires further investigation, here I postulate that the presence of autoimmunity to cartilage components has an important role in the process of cartilage degradation in OA. Current studies strongly suggest that a immunoregulatory therapeutic strategy should be established.     

Autoimmune response in cartilage-delivered peptides in a patient with osteoarthritis

Whatever the initiating factor of osteoarthritis (OA), the process ultimately unmasks the immunogenic determinants of chondrocytes, proteoglycans and collagens, which then triggers autoimmune reactions. Although the precise mechanism of the immune responses in the pathogenesis of OA requires further investigation, here I postulate that the presence of autoimmunity to cartilage components has an important role in the process of cartilage degradation in OA. Current studies strongly suggest that a immunoregulatory therapeutic strategy should be established.     

Biochemical analysis of normal and osteoarthritic human cartilage

Non-collagenous proteins from the articular cartilage of normal subjects and patients with degenerative joint disease were extracted sequentially. Proteoglycans and the other glycoproteins were more extractable from the osteoarthritic cartilage at lower ionic strength than those from the normal cartilage. A 50-kD protein which seems specific to osteoarthritic cartilage was identified. Three different populations of proteoglycans were purified from normal and only two from osteoarthritic cartilage. Moreover, greater amounts of albumin and fibronectin were found in the pathological cartilage. No differences were observed between link proteins from normal and osteoarthritic cartilage, nor in their molecular weight or the amounts extracted.     

Never say never again: protein glycosylation in pathogenic bacteria

In recent years, accumulating evidence for glycosylated bacterial proteins has overthrown an almost dogmatic belief that prokaryotes are not able to synthesize glycoproteins. Now it is widely accepted that eubacteria express glycoproteins. Although, at present, detailed information about glycosylation and structure-function relationships is available for only few eubacterial proteins, the variety of different components and structures observed already indicates that the variations in bacterial glycoproteins seem to exceed the rather limited display found in eukaryotes. Numerous virulence factors of bacterial pathogens have been found to be covalently modified with carbohydrate residues, thereby identifying these factors as true glycoproteins. In several bacterial species, gene clusters suggested to represent a general protein glycosylation system have been identified. In other cases, genes encoding highly specific glycosyltransferases have been found to be directly linked with virulence genes. These findings raise interesting questions concerning a potential role of glycosylation in pathogenesis. In this review, we will therefore focus on protein glycosylation in Gram-negative bacterial pathogens.     

Fast-Transported Glycoproteins and Nonglycosylated Proteins Contain Sulfate

35SO4-labeled fast-transported proteins of bullfrog dorsal root ganglion neurons were separated by two-dimensional gel electrophoresis, and their mobilities were compared to similar species labeled with [3H]mannose or [3H]fucose. Fluorography revealed regions of poorly resolved, high molecular weight material, likely to represent sulfated proteoglycans, as well as many well resolved spots that corresponded in mobility to individual [35S]methionine-labeled fast-transported proteins. The majority of these well resolved spots appeared as "families," previously identified as glycoproteins based on their labeling with sugars. Thus, sulfate can be a contributor to the carbohydrate side-chain charge that underlies microheterogeneity. The most heavily 35SO4-labeled species, however, corresponded to fast-transported proteins that were not labeled by either sugar. The relative acid labilities of 35SO4 associated with individual species cut from the gel confirmed the assignments of these spots as glycoproteins or nonglycoproteins. A group of spots intermediate in their acid lability was also detected, suggesting that some proteins may contain sulfate linked to carbohydrate as well as to amino acid residues.     

Glycoproteins and glycosaminoglycans synthesized by human keratinocytes in culture. Their role in cell-substratum adhesion.

Glycoproteins and proteoglycans synthesized by human keratinocytes in medium containing D-[1-14C]glucosamine were extracted and analysed by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Extraction of the labelled keratinocytes with 0.5% Triton X-100 removed most of the glycoconjugates and left the cytoskeleton and nuclear residue adherent to the substratum. In addition to the cytoskeletal proteins, there was a relatively simple profile of glycoproteins and glycosaminoglycans associated with this adherent cytoskeleton. These consisted of eight glycoproteins in the mol.wt. range 99000-232000, five proteins in the keratin region (mol.wt. 42000-61000), hyaluronic acid and a sulphated glycosaminoglycan. Surface labelling of the keratinocytes with galactose oxidase (with or without neuraminidase)/KB3H4 revealed that many of the glycoproteins were exposed on the cell surface. The importance of the glycoproteins and proteoglycans in attaching the keratinocytes to the substratum was examined by studying their expression after incubation in medium containing tunicamycin and their degradation after digestion with trypsin and hyaluronidase. These studies, together with an examination of the glycoconjugates released by sequential extraction with 0.5% Triton X-100 followed by 0.2% sodium dodecyl sulphate, revealed that the glycoprotein of mol.wt. 232000 has an important role in mediating the attachment of keratinocytes to the substratum.     

Mechanisms of chain initiation in the biosynthesis of connective tissue polysaccharides

Carbohydrate-protein linkages of three types are found in the connective tissue proteoglycans; these linkages involve the following monosaccharide-amino acid pairs: xylose-serine; N-acetylglucosamine-asparagine; and N-acetylgalactosamine-threonine (or serine). The biosynthesis of carbohydrate groups containing linkages of the latter two types presumably occurs by the same pathways that have been well established for many glycoproteins, but details of these processes as they pertain to proteoglycans are not yet known. Initiation of polysaccharide chains linked by the xylose-serine linkage takes place by direct transfer of xylose from UDP-xylose to the hydroxyl groups of specific serine residues in the core proteins of the respective proteoglycans, and the xylosyltransferase catalyzing these reactions has been detected in the rough endoplasmic reticulum of embryonic chick chondrocytes. Although the completed or nascent core proteins are the natural substrates for xylose transfer in the intracellular assembly of proteoglycans, a survey of potential exogenous substrates has shown that small peptides containing alternating serine and glycine residues may also serve as acceptors in this reaction. Nevertheless, larger substrates are preferred, such as chondroitin sulfate proteoglycan, which has been deglycosylated by Smith degradation or HF treatment, or silk fibroin, which contains Ser-Gly pairs. In contrast to the sulfated polysaccharides, which are synthesized by carbohydrate transfer to protein in the endoplasmic reticulum and the Golgi apparatus, hyaluronic acid is formed in the plasma membrane by a different mechanism. The reaction by which chains are initiated is not yet known, but recent work by Prehm suggests that this process occurs either by transfer of the glucuronosyl component of UDP-glucuronic acid to UDP-N-acetylglucosamine or by the converse reaction, i.e., transfer of the N-acetylglucosaminyl unit of UDP-N-acetylglucosamine to UDP-glucuronic acid.     

Isolation and characterization of two glycoproteins from hyaline cartilage

Two acidic glycoproteins of molecular mass 92 kDa and 56 kDa were purified from 4 M guanidine hydrochloride extracts of chick sternal cartilage, by density gradient centrifugation, ion-exchange chromatography, gel chromatography and SDS/PAGE. The glycoproteins differed in their amino acid and carbohydrate compositions. They were identified by the immunoblotting technique in extracts of chick articular cartilage from various sites and in extracts of cartilage from other species. The proteins are synthesized by the chondrocytes and show a partial cross-reactivity between their antisera.     

Chondrodysplasias due to proteoglycan defects

The proteoglycans, especially the large chondroitin sulfate proteoglycan aggrecan, have long been viewed as important components of the extracellular matrix of cartilage. The drastic change in expression during differentiation from mesenchyme to cartilage, the loss of tissue integrity associated with proteoglycan degradation in several disease processes and, most important, the demonstration of abnormalities in proteoglycan production concomitant with the aberrant growth patterns exhibited by the brachymorphic mouse, the cartilage matrix deficient mouse, and the nanomelic chick provide the strongest evidence that the proteoglycan aggrecan is essential during differentiation and for maintenance of the skeletal elements. More recently, mutations associated with proteoglycans other than aggrecan, especially the heparan sulfate proteoglycans, glypican and perlecan, suggest an important role for these molecules in skeletal development as well. This review focuses on the molecular bases of the hereditary proteoglycan defects in animal models, as well as of some human chondrodysplasias, that collectively are providing a better understanding of the role of proteoglycans in the development and maintenance of the skeletal elements.     

Histochemistry of mucosubstances in Brunner's-gland cells and duodenal goblet cells of two New World monkey species (Saimiri sciureus and Saguinus fuscicollis)

Duodenal goblet cells and Brunner's-gland cells obtained from two species of New World monkeys (Saimiri sciureus and Saguinus fuscicollis) were studied using conventional histochemical methods and by applying a panel of 17 labelled lectins. The secretions of both goblet and Brunner's-gland cells were found to contain neutral mucosubstances, while those of goblet cells also exhibit acid and sulphated carbohydrate components. Lectin binding studies allowed a more detailed analysis of the mucus glycoproteins. Marked differences between the two examined species were not detected. N-Acetyl-galactosamine, galactose, fucose and N-Acetyl-glucosamine were found to be the predominant sugar residues in Brunner's-glands glycoproteins, with mannose and glucose being only minor components.     

Short review. Inverse relationship between hyaluronan and collagens in development and angiogenesis

Abstract. The extracellular matrix plays a vital role in regulating normal tissue development and function - largely via the specific arrangement of macromolecules such as collagens, proteoglycans, glycosaminoglycans and glycoproteins. Previous reports have concentrated on associations between combinations of collagens/proteoglycans, collagens/glycoproteins and proteoglycans/glycosaminoglycans whilst little information is available on associations between collagens and free glycosaminoglycans.
In this review, we discuss possible associations between collagens and the glycosaminoglycan hyaluronan; macromolecules which are known to exhibit changes in amount and composition during development and under pathological conditions. We demonstrate two types of collagen/hyaluronan association in vivo: the first, during the formation of extracellular matrix structures where neither collagens nor hyaluronan are degraded, resulting in the regulation of collagen fibrillogenesis, and the second, involving an inverse correlation between collagen synthesis and hyaluronan degradation and vice versa. We suggest that associations between collagens and hyaluronan play an important role in the initiation and maintenance of angiogenesis and put forward a model of cartilage vascularisation which relies on these associations.     

Inverse relationship between hyaluronan and collagens in development and angiogenesis

Abstract. The extracellular matrix plays a vital role in regulating normal tissue development and function - largely via the specific arrangement of macromolecules such as collagens, proteoglycans, glycosaminoglycans and glycoproteins. Previous reports have concentrated on associations between combinations of collagens/proteoglycans, collagens/glycoproteins and proteoglycans/glycosaminoglycans whilst little information is available on associations between collagens and free glycosaminoglycans.
In this review, we discuss possible associations between collagens and the glycosaminoglycan hyaluronan; macromolecules which are known to exhibit changes in amount and composition during development and under pathological conditions. We demonstrate two types of collagen/hyaluronan association in vivo: the first, during the formation of extracellular matrix structures where neither collagens nor hyaluronan are degraded, resulting in the regulation of collagen fibrillogenesis, and the second, involving an inverse correlation between collagen synthesis and hyaluronan degradation and vice versa. We suggest that associations between collagens and hyaluronan play an important role in the initiation and maintenance of angiogenesis and put forward a model of cartilage vascularisation which relies on these associations.     

Characterisation of the Carbohydrate Fraction of the Temporary Adhesive Secreted by the Tube Feet of the Sea Star <Emphasis Type="Italic">Asterias rubens</Emphasis>

In sea stars, adhesion takes place at the level of a multitude of small appendages, the tube feet. It involves the secretion of an adhesive material which, after tube foot detachment, remains on the substratum as a footprint. It was previously reported that the two main organic components of this material are proteins and carbohydrates. The carbohydrate moiety of the adhesive secretion of Asterias rubens was investigated using a set of 16 lectins which were used on sections through tube feet, on footprints, and on the proteins extracted from these footprints. After gel electrophoresis, these proteins separate into eight protein bands which were named sea star footprint proteins (Sfps). Eleven lectins label the tube foot epidermis at the level of the adhesive cells, four react with footprints, and eight with two of the extracted footprint proteins, which are therefore classified as glycoproteins. Sfp-290 appears to bear mostly N-linked oligosaccharides and Sfp-210 principally O-linked oligosaccharides. The outer chains of both glycoproteins enclose galactose, N-acetylgalactosamine, fucose, and sialic acid residues. Another part of the carbohydrate fraction of the footprints would be in the form of larger molecules, such as sialylated proteoglycans. These two types of glycoconjugates are presumably key components of the sea star temporary adhesive providing both cohesive and adhesive contributions through electrostatic interactions by the polar and hydrogen-bonding functional groups of their glycan chains.     

Polydispersity and heterogeneity of squid cranial cartilage proteoglycans as assessed by immunochemical methods and electron microscopy

The three populations of squid cranial cartilage proteoglycans, D1D1A, D1D1B and D1D2 appeared to have a high degree of polydispersity. Gel electrophoresis and immunoblotting analysis showed that polydispersity was mainly due to the variable size of chondroitin sulphate E chains. This was further ascertained after rotary shadowing electron microscopy of proteoglycan core proteins and glycosaminoglycan side chains and statistical analysis of the sizes measured for both components. Enzymic treatment of the proteoglycan core proteins produced different peptides from each population, suggesting that the observed heterogeneity of the proteoglycans is due to their core proteins. Antibodies were raised in rabbits against all proteoglycans and enzyme-linked immunosorbent analysis of proteoglycan core proteins revealed that the proteoglycans, even heterogeneous, shared many common epitopes. Part of the common proteoglycan epitopes were found to be located in chondroitin sulphate E chains. Heterogeneity of squid proteoglycans was also investigated by studying their interactions with collagen and it was found that only the two populations of high molecular mass, D1D1A and D1D2, were able to interact with only collagen type I, the latter stronger than the former.     

细胞外基质与基质金属蛋白酶

细胞外基质（ECM）是存在于细胞之间的动态网状结构,由胶原、蛋白聚糖及糖蛋白等大分子物质组成.这些大分子物质可与细胞表面上的特异性受体结合,通过受体与细胞骨架结构直接发生联系或触发细胞内的一系列信号传导而引起不同的基因表达,从而导致细胞的生长和分化.作为降解ECM成分最重要的酶-基质金属蛋白酶（MMPs）及其组织抑制因子(TIMPs)在这一过程中起着非常重要的作用.MMPs是一类依赖金属离子锌并以ECM成分为水解底物的蛋白水解酶.其在转录水平的表达受到生长因子、细胞因子及激素等因素的严格调控,在蛋白质水平其活性也受到其激活剂和抑制剂的调节. MMPs通过对ECM成分的水解来影响其降解与重组的动态平衡而参与多种细胞的生理和病理过程.     

Tumour-associated and differentiation antigens on the carbohydrate moieties of mucin-type glycoproteins

In this report the carbohydrate antigens expressed on the three oligosaccharide domains, core, backbone and peripheral, of mucin-type glycoproteins are briefly reviewed in the light of recent observations with monoclonal antibodies. These have revealed that a number of cell-surface antigens which behave as tumour-associated and differentiation antigens of man or mouse are abundantly expressed on the carbohydrate chains of a variety of secreted mucins of human and animal origins and they belong to an antigen system which also includes the major blood group antigens. Examples are given of the use of well-characterized anti-carbohydrate antibodies to derive structural information on (a) mucin-type glycoproteins of human B lymphocyte membranes, (b) the high molecular weight glycoproteins of the normal human gastric and distal-colon mucosae and (c) tumour-derived glycoproteins from these two organs. Major differences between the antigenicities of the normal stomach and distal-colon, and between their tumour-derived glycoproteins, and the important effect of the secretor status in the expression of these antigens are described. These observations have enabled a better understanding of the individual and tissue differences in the expression of tumour-associated antigens. The possibility is raised that these carbohydrate structures (many of which also occur on certain N-linked oligosaccharides and glycolipids) are components of receptor systems for endogenous ligands. More tangible evidence is cited for the role of certain structures in this family of saccharides as receptors for infective agents.     

The role of N-linked oligosaccharides in glycoprotein function.

N-linked glycoproteins include such biologically important molecules as cell-surface receptors, cell-adhesion molecules, immunoglobulins and other serum proteins, and tumor antigens. Investigating the role of carbohydrate in glycoprotein function has included the use of glycosylation inhibitors or site-directed mutagenesis of specific glycosylation sites to prevent the addition of carbohydrate, or glycosylation processing inhibitors or animal cell glycosylation mutants to alter carbohydrate structure. In some proteins, glycosylation plays an important role in recognition, while in others, it may stabilize and/or control the conformation of the protein. The cloning of genes in bacteria or lower eukaryotes--with the goal of producing biologically active proteins for biotechnological purposes--necessitates a better understanding of the role of specific carbohydrate structures.