Different usage of the glycosaminoglycan attachment sites of biglycan

Biglycan is a member of the small leucine-rich proteoglycan family. Its core protein comprises two chondroitin/dermatan sulfate attachment sites on serine 42 and serine 47, respectively, which are the fifth and tenth amino acid residues, respectively, after removal of the prepro peptide. Because the regulation of glycosaminoglycan chain assembly is not fully understood and because of the in vivo existence of monoglycanated biglycan, mutant core proteins were stably expressed in human 293 and Chinese hamster ovary cells in which i) either one or both serine residues were converted into alanine or threonine residues, ii) the number of acidic amino acids N-terminal of the respective serine residues was altered, and iii) a hexapeptide was inserted between the mutated site 1 and the unaltered site 2. Labeling experiments with [(35)S]sulfate and [(35)S]methionine indicated that serine 42 was almost fully used as the glycosaminoglycan attachment site regardless of whether site 2 was available or not for chain assembly. In contrast, substitution of site 2 was greatly influenced by the presence or absence of serine 42, although additional mutations demonstrated a direct influence of the amino acid sequence between the two sites. When site 2 was not substituted with a glycosaminoglycan chain, there was also no assembly of the linkage region. These results indicate that xylosyltransferase is the rate-limiting enzyme in glycosaminoglycan chain assembly and implicate a cooperative effect on the xylosyl transfer to site 2 by xylosylation of site 1, which probably becomes manifest before the removal of the propeptide. It is shown additionally that biglycan expressed in 293 cells may still contain the propeptide sequence and may carry heparan sulfate chains as well as sulfated N-linked oligosaccharides.     

Isolation and sequence analysis of the glycosaminoglycan attachment site of type IX collagen

Type IX collagen from chick embryonic cartilage is unique among the collagens in that it contains chondroitin sulfate covalently linked to the alpha 2(IX) polypeptide chain. We have isolated and sequenced the glycosaminoglycan-containing peptide released by collagenase digestion from type IX collagen, labeled biosynthetically with [35SO4] and 3H-aminoacids. This peptide was purified by gel filtration and, following chondroitinase ABC digestion, by reverse-phase high performance liquid chromatography. The amino acid sequence obtained for this peptide has 23 residues, beginning and ending with a collagenous sequence, indicating that it spans an internal noncollagenous domain. Comparison of this sequence with the one predicted from cDNA clone pYN 1738 for the alpha 1(IX)chain and pYN 1731 and pDM 222 for the alpha 2(IX)chain revealed the peptide to be the noncollagenous NC3 domain of alpha 2(IX). The glycosylated sequence Val-Glu-Gly-Ser*-Ala-Asp- of type IX collagen does not have the Ser-Gly normally functioning as the attachment sequence but does have an acidic residue preceding the serine which should improve the acceptability of this sequence for the xylosyltransferase. That it is an adequate acceptor can be inferred from the observation that type IX collagen carries a glycosaminoglycan chain on over 70% of the molecules isolated.     

Fluorescent localization of calcium at sites of cell attachment and spreading

Chlorotetracycline (CTC), a calcium-chelating fluorescent probe identifies and localizes calcium when applied to cultured neural crest cells. The fluorescence occurs at specific membrane regions involved in cell attachment and spreading as well as with the formation of cytoskeletal stress fibers (actin microfilament bundles). The observed CTC reaction indicates that calcium sequestration and the development of these membrane-cytoskeletal features share a temporal and spatial pattern. Thus, the selective availability of calcium may be an important determinant of cell morphogenesis.     

The glycosaminoglycan attachment regions of human aggrecan

Aggrecan possesses both chondroitin sulfate (CS) and keratan sulfate (KS) chains attached to its core protein, which reside mainly in the central region of the molecule termed the glycosaminoglycan-attachment region. This region is further subdivided into the KS-rich domain and two adjacent CS-rich domains (CS1 and CS2). The CS1 domain of the human is unique in exhibiting length polymorphism due to a variable number of tandem amino acid repeats. The focus of this work was to determine how length polymorphism affects the structure of the CS1 domain and whether CS and KS chains can coexist in the different glycosaminoglycan-attachment domains. The CS1 domain possesses several amino acid repeat sequences that divide it into three subdomains. Variation in repeat number may occur in any of these domains, with the consequence that CS1 domains of the same length may possess different amino acid sequences. There was no evidence to support the presence of KS in either the CS1 or the CS2 domains nor the presence of CS in the KS-rich domain. The structure of the CS chains was shown to vary between the CS1 and CS2 domains, particularly in the adult, with variation occurring in chain length and the sulfation of the non-reducing terminal N-acetyl galactosamine residue. CS chains in the adult CS2 domain were shorter than those in the CS1 domain and possessed disulfated terminal residues in addition to monosulfated residues. There was, however, no change in the sulfation pattern of the disaccharide repeats in the CS chains from the two domains.     

Carriers for enzymatic attachment of glycosaminoglycan chains to peptide

In the previous study, we have found that the endo-beta-xylosidase from Patinopecten had the attachment activities of glycosaminoglycan (GAG) chains to peptide. As artificial carrier substrates for this reaction, synthesis of various GAG chains having the linkage region tetrasaccharide, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl, between GAG chain and core protein of proteoglycan was investigated. Hyaluronic acid (HA), chondroitin (Ch), chondroitin 4-sulfate (Ch4S), chondroitin 6-sulfate (Ch6S), and desulfated dermatan sulfate (desulfated DS) as donors and the 4-metylumbelliferone (MU)-labeled hexasaccharide having the linkage region tetrasaccharide at its reducing terminals (MU-hexasaccharide) as an acceptor were subjected to a transglycosylation reaction of testicular hyaluronidase. The products were analyzed by high-performance liquid chromatography and enzyme digestion, and the results indicated that HA, Ch, Ch4S, Ch6S, and desulfated DS chains elongated by the addition of disaccharide units to the nonreducing terminal of MU-hexasaccharide. It was possible to custom-synthesize various GAG chains having the linkage region tetrasaccharide as carrier substrates for enzymatic attachment of GAG chains to peptide.     

The multiple functions of collagen XVIII in development and disease

Collagen XVIII is a heparan sulphate proteoglycan which is expressed ubiquitously in different basement membranes throughout the body. Its C-terminal fragment, endostatin, has been found to inhibit angiogenesis and tumor growth by restricting endothelial proliferation and migration and inducing apoptosis of endothelial cells. Collagen XVIII has three variants, of which the shortest one is found in most vascular and epithelial BM structures, whereas the longer variants are found especially in the liver. The longest or frizzled variant has a cysteine-rich domain in its N-terminus that has been shown to inhibit Wnt signaling in vitro. The presence of collagen XVIII homologues in organisms such as C. elegans, Xenopus laevis, zebrafish and chick suggests a fundamental role for this BM collagen. Mutations in the collagen XVIII gene lead to the Knobloch syndrome, which is characterized by high myopia, vitreoretinal degeneration with retinal detachment, macular abnormalities and occipital encephalocele. Mice lacking collagen XVIII also show several ocular abnormalities. This suggests that in physiological conditions collagen XVIII is mostly needed for the proper development of the eye. Moreover, it appears to be needed for the structural stability of basement membranes in several other organs, and increasing evidence shows its importance for other organs in non-physiological situations such as atherosclerosis, glomerulonephritis or other type of tissue damage. This review focuses on clarifying the roles of collagen XVIII and its variants and domains in various physiological and pathological conditions.     

Culturing chick muscle cells on glycosaminoglycan substrates: attachment and differentiation

The effects of different glycosaminoglycans (GAGs) on myogenesis were tested by culturing embryonic chick myoblasts on tissue culture dishes to which either hyaluronic acid (HA) or chondroitin sulfate (ChS) was covalently bound. Both in cell number and in apparent cell type distribution, the population of cells bound to GAGs is similar to that on gelatin and significantly different from that observed with uncoated dishes. When plated on ChS, myoblasts proliferate, align, and fuse at a rate similar to cells plated on gelatin. The final fused cells appear as sheets rather than long, thin myotubes. On HA, the cells proliferate but are inhibited from differentiation. The extent of inhibition is dependent on the amount of HA present. The inhibition of myogenesis is maintained through four subcultures on HA, but can be reversed at any time by culturing cells on gelatin. These experiments indicate that different GAGs have different effects on myogenesis and that HA can actively inhibit the process.     

Lack of collagen XVIII/endostatin results in eye abnormalities

Mice lacking collagen XVIII and its proteolytically derived product endostatin show delayed regression of blood vessels in the vitreous along the surface of the retina after birth and lack of or abnormal outgrowth of retinal vessels. This suggests that collagen XVIII/endostatin is critical for normal blood vessel formation in the eye. All basement membranes in wild-type eyes, except Descemet's membrane, showed immunogold labeling with antibodies against collagen XVIII. Labeling at sites where collagen fibrils in the vitreous are connected with the inner limiting membrane and separation of the vitreal matrix from the inner limiting membrane in mutant mice indicate that collagen XVIII is important for anchoring vitreal collagen fibrils to the inner limiting membrane. The findings provide an explanation for high myopia, vitreoretinal degeneration and retinal detachment seen in patients with Knobloch syndrome caused by loss-of-function mutations in collagen XVIII.     

Cytochemical localization of DNA loop attachment sites to the nuclear lamina and to the inner nuclear matrix

Summary The rat liver nuclear matrix, obtained by endogenous nuclease digestion and extraction with low and high lonic strength media, contains residual DNA fragments that are considered to represent the attachment sites of the chromatin domains to the nucleoskeleton. These sites, protected against nuclease digestion by their binding with the nucleoskeleton proteins, should be either mainly linked to the peripheral lamina or to the inner nuclear matrix. The DNA fragment distribution at the level of the different components of the nuclear matrix has been evaluated in samples embedded in Epon and in hydrophilic resins by means of the DNase-gold technique. The labeling obtained suggests that the chromatin loops are prevailingly associated with the interior of the matrix; in fact about twice of the label is present in the inner matrix with respect to the peripheral lamina area.These results confirm the hypothesis that in interphase the chromatin maintains an organization similar to that of chromosomes, with loops radiating from a central scaffold, instead of being mainly attached to the lamina as otherwise suggested.     

Laminin modulates morphogenic properties of the collagen XVIII endostatin domain

We have shown previously that the oligomeric endostatin domain of collagen XVIII (NC1) functioned as a motility-inducing factor regulating the extracellular matrix-dependent morphogenesis of endothelial cells. This motogenic activity gave rise to structures resembling filipodia and lamellipodia and was dependent on Rac, Cdc42, and mitogen-activated protein kinase. Here, we demonstrate that these properties of endostatin are primarily mediated by laminin in the basement membrane and heparan sulfates on the cell surface. The sites of interaction between laminin and oligomeric endostain include the N-terminal regions of all three laminin chains (amino acids 204-1243 of the alpha chain, 932-1161 of the beta chain, and 150-965 of the gamma chain). A monoclonal antibody that blocks the interactions between endostatin and laminin was utilized to inhibit the motogenic activity of endostatin. In parallel, we have engineered selective point mutations and produced recombinant forms that lack binding to heparan sulfates on the cell surface. Our data are consistent with a model of endostatin with two binding sites: one mainly to laminin in the basement membrane and the other to heparan sulfates on the cell surface. The two binding domains on endostatin appear to be separate with the possibility of some overlap between the two sites.     

Replication origins, factors and attachment sites

The initiation of eukaryotic DNA synthesis occurs at specific sites determined by both cis- and trans-acting elements. Here I review advances in the characterization of yeast origins, origin-binding proteins and the relationship of DNA replication to nuclear substructure in yeast.     

Identification of L-selectin binding heparan sulfates attached to collagen type XVIII

L-selectin is a C-type lectin expressed on leukocytes that is involved in both lymphocyte homing to the lymph node and leukocyte extravasation during inflammation. Known L-selectin ligands include sulfated Lewis-type carbohydrates, glycolipids, and proteoglycans. Previously, we have shown that in situ detection of different types of L-selectin ligands is highly dependent on the tissue fixation protocol used. Here we use this knowledge to specifically examine the expression of L-selectin binding proteoglycans in normal mouse tissues. We show that L-selectin binding chondroitin/dermatan sulfate proteoglycans are present in cartilage, whereas L-selectin binding heparan sulfate proteoglycans are present in spleen and kidney. Furthermore, we show that L-selectin only binds a subset of renal heparan sulfates, attached to a collagen type XVIII protein backbone and predominantly present in medullary tubular and vascular basement membranes. As L-selectin does not bind other renal heparan sulfate proteoglycans such as perlecan, agrin, and syndecan-4, and not all collagen type XVIII expressed in the kidney binds L-selectin, this indicates that there is a specific L-selectin binding domain on heparan sulfate glycosaminoglycan chains. Using an in vitro L-selectin binding assay, we studied the contribution of N-sulfation, O-sulfation, C5-epimerization, unsubstituted glucosamine residues, and chain length in L-selectin binding to heparan sulfate/heparin glycosaminoglycan chains. Based on our results and the accepted model of heparan sulfate domain organization, we propose a model for the interaction of L-selectin with heparan sulfate glycosaminoglycan chains. Interestingly, this opens the possibility of active regulation of L-selectin binding to heparan sulfate proteoglycans, e.g. under inflammatory conditions.     

Phenothiazine-binding and attachment sites of CAPP1-calmodulin

In the presence of Ca2+ norchlorpromazine isothiocyanate forms a monocovalent complex with calmodulin: CAPP1-calmodulin (Newton et al, 1983). Trypsin digestion of [3H]CAPP1-calmodulin yields as the major radioactive peptide N epsilon-CAPP-Lys-Met-Lys, corresponding to residues 75-77 of calmodulin. Stoichiometric amounts of all other expected tryptic peptides are also found, indicating that norchlorpromazine isothiocyanate selectively acylates Lys 75. A second molecule of CAPP-NCS can react, albeit slowly, with calmodulin to form CAPP2-calmodulin. Fragments 38-74 and 127-148 are completely missing from the trypsin digests of CAPP2-calmodulin without deliberate exposure to UV irradiation. Possibly the lengthy preparation of CAPP2-calmodulin favors photolysis, caused by room lights, of the putative CAPP-binding domains located in these two peptides. Lys 148, the sole lysyl residue in fragment 127-148, is a probable site of attachment of the second molecule of CAPP. UV irradiation of CAPP1-calmodulin, followed by digestion with trypsin, results in the selective loss of 50% each of peptides containing residues 38-74 and 127-148, suggesting that these peptides contain the hydrophobic amino acids that form the phenothiazine-binding sites. The loss of peptides encompassing residues 38-74 and 127-148, located in the amino and carboxyl halves of calmodulin, respectively, suggests that the hydrophobic rings of CAPP can bind at either one of the two phenothiazine sites. Computer modeling of CAPP1-calmodulin with the X-ray coordinates of calmodulin (Babu et al., 1986) indicates that CAPP attached to Lys 75 cannot interact with the carboxyl-terminal phenothiazine-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of collagen XVIII accelerates cutaneous wound healing, while overexpression of its endostatin domain leads to delayed healing

Endostatin, the C-terminal fragment of collagen XVIII, is known to suppress tumour growth and angiogenesis by inhibiting endothelial cell proliferation and migration. We have previously shown that endostatin and its precursor are important for the structural organization of basement membranes (BM). The aim of this study was to investigate cutaneous wound healing in mice overexpressing endostatin in keratinocytes (ES-tg) and in mice lacking collagen XVIII (Col18a1(-/-)). Excisional wounds were made on the dorsal skin of mice, the wound areas were measured and the wounds were collected for further analyses after 3, 6 or 14 days. The healing of the wounds was delayed in the ES-tg mice and accelerated in the Col18a1(-/-) mice, and the vascularisation rate was accelerated in the Col18a1(-/-) mice, but not affected in the ES-tg mice. Abnormal capillaries with swollen endothelial cells and narrowed lumens were observed in the wounds of the ES-tg mice. In these mice also the formation of the epidermal BM was delayed, and the structure of the epidermal and capillary BMs was more disorganised. Moreover, detachment of the epidermis from the granulation tissue was observed in half (n=10) of the 6-day-old ES-tg wounds, but in none of the controls, suggesting an increased fragility of the epidermal-dermal junction in the presence of an excess of endostatin.     

Comparison of collagen and glycosaminoglycan synthesis in attaching control and diabetic human skin fibroblasts

Summary Cultured fibroblasts derived from normal subjects and juvenile diabetics attach in the absence of serum to plastic culture dishes and secrete macromolecules, including collagenous components, hyaluronic acid, and proteoglycans into the medium and onto the plastic surface where they form a microexudate carpet. Most diabetic fibroblasts examined did not spread as well as normal cells during a 4-hr interval after the initial attachment. There were no significant differences between normal and diabetic cells with respect to proline and lysine incorporation and lysine hydroxylation. The percentage glycosylation of hydroxylysine was marginally higher in the media proteins of diabetic cells, but glycosylation in both normal and diabetic cells was elevated over that typically observed in human skin collagen. Collagenous components were estimated to constitute approximately 15–20% of the microexudate carpet fraction in both normal and diabetic cell strains. Diabetic fibroblasts exhibited a marginally lower ratio of heparan sulfate to chondroitin sulfate in the cell surface to matrix microexudate carpet fraction (trypsinate) than did normal fibroblasts. The hyaluronate and chondroitin sulfate contents of this fraction of diabetic cells were not significantly different from those of normal cells. This work was supported by Grants AM 11821 and AM 19606 from the National Institutes of Health, United States Public Health Service, by a grant from the American Diabetes Association (Leon W. Cunningham, Principal Investigator) and by the Vanderbilt Diabetes Endocrinology Center grant, AM 17026. R. E. Branson was the recipient of a research fellowship of the Juvenile Diabetes Foundation and of a National Institutes of Health Postdoctoral Fellowship AM 05636.     

Localization of glycosaminoglycan substitution sites on domain V of mouse perlecan

Perlecan, the predominant basement membrane proteoglycan, has previously been shown to contain glycosaminoglycans attached at serine residues, numbers 65, 71, and 76, in domain I. However, the C-terminal domains IV and V of this molecule may also be substituted with glycosaminoglycan chains, but the exact substitution sites were not identified. The amino acid sequence of mouse perlecan reveals many ser-gly sequences in these domains that are possible sites for glycosaminoglycan substitution. We expressed recombinant domain IV and/or V of mouse perlecan in COS-7 cells and analyzed glycosaminoglycan substitution. Both heparan sulfate and chondroitin sulfate chains could be detected on recombinant domain V. One site, ser-gly-glu (serine residue 3593), toward the C-terminal region of domain V is a substitution site for heparan sulfate. When this sequence was absent, chondroitin/dermatan sulfate substitution was deleted, and the likely site for this galactosaminoglycan substitution was ser-gly-ala-gly (serine residue 3250) on domain V.     

Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins

Although interactions of proteins with glycosaminoglycans (GAGs), such as heparin and heparan sulphate, are of great biological importance, structural requirements for protein-GAG binding have not been well-characterised. Ionic interactions are important in promoting protein-GAG binding. Polyelectrolyte theory suggests that much of the free energy of binding comes from entropically favourable release of cations from GAG chains. Despite their identical charges, arginine residues bind more tightly to GAGs than lysine residues. The spacing of these residues may determine protein-GAG affinity and specificity. Consensus sequences such as XBBBXXBX, XBBXBX and a critical 20 Å spacing of basic residues are found in some protein sites that bind GAG. A new consensus sequence TXXBXXTBXXXTBB is described, where turns bring basic interacting amino acid residues into proximity. Clearly, protein-GAG interactions play a prominent role in cell-cell interaction and cell growth. Pathogens including virus particles might target GAG-binding sites in envelope proteins leading to infection. BioEssays 20:156–167, 1998. © 1998 John Wiley & Sons, Inc.