scholarly journals Glycosylation of CD44 is implicated in CD44-mediated cell adhesion to hyaluronan.

1996 ◽  
Vol 132 (6) ◽  
pp. 1199-1208 ◽  
Author(s):  
A Bartolazzi ◽  
A Nocks ◽  
A Aruffo ◽  
F Spring ◽  
I Stamenkovic
Keyword(s):  
Cell Adhesion ◽  
Regulatory Mechanism ◽  
Cell Attachment ◽  
Glycosylation Site ◽  
Side Chain ◽  
Site Specific ◽  
Coated Substrate ◽  
Glycosylation Sites ◽  
Coated Surfaces

CD44-mediated cell adhesion to hyaluronate is controlled by mechanisms which are poorly understood. In the present work we examine the role of N-linked glycosylation and Ser-Gly motifs in regulating CD44-hyaluronate interaction. Our results show that treatment of a panel of human cell lines which constitutively express CD44 with the inhibitor of N-linked glycosylation tunicamycin results in the loss of attachment of these cells to hyaluronate-coated substrate. In contrast, treatment of the same cells with deoxymannojirimycin, which inhibits the conversion of high mannose oligosaccharides to complex N-linked carbohydrates, results in either no change or an increase in CD44-mediated adhesion to hyaluronate, suggesting that complex N-linked oligosaccharides may not be required for and may even inhibit CD44-HA interaction. Using human melanoma cells stably transfected with CD44 N-linked glycosylation site-specific mutants, we show that integrity of five potential N-linked glycosylation sites within the hyaluronate recognition domain of CD44 is critical for hyaluronate binding. Mutation of any one of these potential N-linked glycosylation sites abrogates CD44-mediated melanoma cell attachment to hyaluronate-coated surfaces, suggesting that all five sites are necessary to maintain the HA-recognition domain in the appropriate conformation. We also demonstrate that mutation of serine residues which constitute the four Ser-Gly motifs in the membrane proximal domain, and provide potential sites for glycosaminoglycan side chain attachment, impairs hyaluronate binding. Taken together, these observations indicate that changes in glycosylation of CD44 can have profound effects on its interaction with hyaluronic acid and suggest that glycosylation may provide an important regulatory mechanism of CD44 function.

scholarly journals Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin

2018 ◽  
Vol 475 (9) ◽  
pp. 1583-1595 ◽  
Author(s):  
Midori Oyama ◽  
Yoshinobu Kariya ◽  
Yukiko Kariya ◽  
Kana Matsumoto ◽  
Mayumi Kanno ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Αvβ3 Integrin ◽  
Single Mutation ◽  
Rich Region ◽  
Site Specific ◽  
Phosphorylation Level ◽  
Glycosylation Sites ◽  
Adhesion Activity ◽  
Proline Rich Region

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr134/Thr138/Thr143/Thr147/Thr152) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr134/Thr138 or Thr143/Thr147/Thr152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC–MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation.

scholarly journals Studies on cell adhesion and recognition. II. The kinetics of cell adhesion and cell spreading on surfaces coated with carbohydrate-reactive proteins (glycosidases and lectins) and fibronectin.

1981 ◽  
Vol 88 (1) ◽  
pp. 138-148 ◽  
Author(s):  
W G Carter ◽  
H Rauvala ◽  
S I Hakomori
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Cell Attachment ◽  
Cell Spreading ◽  
Galactose Oxidase ◽  
Cross Linking ◽  
Con A ◽  
Coated Surfaces ◽  
Plastic Surfaces ◽  
Kinetics Of

The kinetics of cell attachment and cell spreading on the coated surfaces of two classes of carbohydrate-reactive proteins, enzymes and lectins, have been compared with those on fibronectin-coated surfaces with the following results: (a) A remarkable similarity between the kinetics of cell attachment to fibronectin-coated and glycosidase-coated surfaces was found. In contrast, cell attachment kinetics induced by lectin- and galactose oxidase-coated surfaces, in general, were strikingly different from those on fibronectin and glycosidase surfaces. The distinction between fibronectin- or glycosidase- and lectin- or galactose oxidase (an enzyme with lectin-type characteristics)-coated surfaces was further supported by the finding that cytochalasin B and EDTA inhibited cell attachment to fibronectin- and glycosidase-coated surfaces but not lectin-coated surfaces. (b) Fibronectin, if labeled and added to a cell suspension, showed only low or negligible interaction with the cell surface. However, fibronectin absorbed on plastic surfaces showed a high cell-attaching activity. It is assumed that fibronectin coated on plastic surfaces may form polyvalent attachment sites in contrast to its lower valency in aqueous solution. (c) Various inhibitors of cell attachment to both fibronectin-, galactose oxidase-, and lectin-coated surfaces were effective only during the first few minutes of the adhesion assay, after which time the attached cells became insensitive to the inhibitors. It is suggested that the initial specific recognition on either lectin-type or fibronectin-type surfaces is followed by an active cell-dependent attachment process. The primary role of the adhesion surface is to stimulate the cell-dependent attachment response. (d) Cells attached on tetravalent concanavalin A (Con A) spread very rapidly and quantitatively, whereas divalent succinyl Con A and monovalent Con A were effective stimulators of cell attachment but not cell spreading. Cross-linking of succinyl Con A restored the cell spreading activity. Tetravalent Con A surfaces specifically bind soluble glycoproteins, whereas succinyl Con A has a greatly reduced ability to bind the same glycoproteins. These results suggest that cross-linking of cell surface glycoproteins by the multivalent adhesive surface may trigger the cellular reaction leading to cell spreading.

Comments on the Clinical Application of Fibronectin in Dentistry

1988 ◽  
Vol 67 (2) ◽  
pp. 515-517 ◽  
Author(s):  
B.S. Pearson ◽  
R.J. Klebe ◽  
B.D. Boyan ◽  
D. Moskowicz
Keyword(s):  
Cell Adhesion ◽  
Oral Surgery ◽  
Cell Attachment ◽  
Root Surface ◽  
Tooth Surface ◽  
Tooth Surfaces ◽  
Tissue Attachment ◽  
Cell Adhesion Proteins ◽  
Demineralized Bone

Several studies have demonstrated that citric acid demineralization of the root surface promotes tissue attachment. Since demineralization exposes collagen to which fibronectin binds, the role of fibronectin in the attachment of cells to the tooth surface has been of considerable interest. It is clear that fibronectin and other cell adhesion proteins can promote cell attachment to the tooth surface; therefore, attempts have been made to utilize these findings in a clinical setting. Using a quantitative ELISA procedure to measure the binding of fibronectin to demineralized bone and tooth, we have found that I μg fibronectin can saturate approximately 1 mg of either demineralized bone or demineralized tooth powder. Since serum contains 300 μg fibronectin per mL, the bleeding that occurs during oral surgery should saturate exposed tooth surfaces with amounts of fibronectin adequate for cell adhesion. Thus, exogenous fibronectin would appear to be of little clinical benefit.

scholarly journals Role of N-glycosylation in the synthesis, dimerization and secretion of human interferon-γ

1994 ◽  
Vol 303 (3) ◽  
pp. 831-840 ◽  
Author(s):  
T Sareneva ◽  
J Pirhonen ◽  
K Cantell ◽  
N Kalkkinen ◽  
I Julkunen
Keyword(s):  
Interferon Γ ◽  
Glycosylation Site ◽  
Biologically Active ◽  
Human Interferon ◽  
Mutant Form ◽  
Ifn Gamma ◽  
Glycosylation Sites ◽  
Mutant Forms ◽  
Kinetics Of

Human interferon-gamma (IFN-gamma) is a secretory glycoprotein, which has two potential N-linked glycosylation sites at positions Asn-25 and Asn-97 of its 143 amino acid long mature polypeptide chain. In order to understand the role of glycan residues in the synthesis and secretion of human IFN-gamma, both or either one of the potential N-linked glycosylation sites were mutated to Gln. The mutant and the wild-type (Wt) polypeptides were expressed in insect cells using a baculovirus vector. Elimination of the N-glycosylation site at position Asn-97 (N97Q) resulted in secreted protein yields of 70-90% as compared with the Wt production, whereas only 10-25% (N25Q) and 1-10% (N25Q,N97Q) levels of protein production was observed when the first or both sites were mutated, respectively. Although there was a difference between extracellular levels of produced protein, the kinetics of secretion was similar for all different IFN-gamma molecules. The Wt and the N-glycosylation site mutants were all secreted as dimers. The formation of biologically active dimers was more efficient for IFN-gamma polypeptides that had the intact glycosylation site at Asn-25 as compared with the other two mutant forms of IFN-gamma. The extent of dimerization correlated well with the observed secretion. The specific antiviral activity was of the same order (1 x 10(7) i.u./mg of protein) for the glycosylated IFN-gamma molecules, whereas it was slightly lower (0.5 x 10(7) i.u./mg of protein) for the unglycosylated mutant form.

scholarly journals Fibronectin glycosylation modulates fibroblast adhesion and spreading.

1986 ◽  
Vol 103 (5) ◽  
pp. 1663-1670 ◽  
Author(s):  
G E Jones ◽  
R G Arumugham ◽  
M L Tanzer
Keyword(s):  
Cell Adhesion ◽  
Biological Functions ◽  
Cell Surfaces ◽  
Fibroblastic Cell ◽  
Cultured Fibroblasts ◽  
Fibroblast Adhesion ◽  
Coated Surfaces ◽  
Composite Data

The role of the carbohydrate residues of fibronectin concerning the specificities of that glycoprotein to interact with fibroblastic cell surfaces, gelatin, and heparin was examined. Tunicamycin was used to produce carbohydrate-depleted fibronectin; it was synthesized by cultured fibroblasts. Unglycosylated and glycosylated fibronectins were analyzed for their ability to bind gelatin and heparin, using affinity columns. Fibronectin-coated surfaces were used to quantitatively measure cell adhesion and spreading. The results showed that the lack of carbohydrates significantly increased the interaction of the protein with gelatin and markedly enhanced its ability to promote adhesion and spreading of fibroblasts. In contrast, the binding of fibronectin to heparin was not influenced by glycosylation. The composite data indicate that the Asn-linked oligosaccharides of fibronectin act as modulators of biological functions of the glycoprotein.

scholarly journals Selective elongation of the oligosaccharide attached to the second potential glycosylation site of yeast exoglucanase: effects on the activity and properties of the enzyme

1994 ◽  
Vol 304 (3) ◽  
pp. 917-922 ◽  
Author(s):  
R D Basco ◽  
L M Hernández ◽  
M D Muñox ◽  
I Olivero ◽  
E Andaluz ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Thermal Inactivation ◽  
Glycosylation Site ◽  
Translation Product ◽  
Site Specific ◽  
Potential Glycosylation Site ◽  
Glycosylation Sites ◽  
Conformational Constraints ◽  
Specific Elongation ◽  
Protein Portion

Three exoglucanases (Exgs), ExgIa, ExgIb and Exg325, are secreted by Saccharomyces cerevisiae cells. They share a common protein portion with two potential glycosylation sites (sequons) but differ in the amount of N-linked carbohydrate [Basco, R.D., Muñoz, M.D., Hernández, L.M., Váquez de Aldana, C. and Larriba, G. (1993) Yeast 9, 221-234]. ExgIb contains two short oligosaccharides attached to asparagines (Asn) 165 and 325 of the primary translation product [Hernández, L.M., Olivero, I., Alvarado, E. and Larriba, G. (1992) Biochemistry 31, 9823-9831]. Exg325 carries a single, short oligosaccharide bound to Asn325 whereas ExgIa has at least one large oligosaccharide, since it has not been produced by mutant mnn9. To address the question of the origin of ExgIa, both sequons were individually mutated by substituting Gln for Asn. An ExgIa-like isoenzyme was still secreted by mutant Exg165 but not by mutant Exg325. Additional studies on sequential deglycosylation of ExgIa with endo-beta-N-acetylglucosaminidase H (endo H), the susceptibility of both oligosaccharides to the endoglycosidase, and analysis of the presence of GlcNAc at both asparagine residues after total deglycosylation with endo H, indicated that ExgIa contained two oligosaccharides, a short one bound to Asn165 and a large one bound to Asn325, and, accordingly, originated from ExgIb. The elongation of the second oligosaccharide did not result in a higher stability towards thermal inactivation or unfolding, or in an increased resistance to proteases as compared with ExgIb; however, the affinity of the enzyme towards laminarin decreased by 50%. This site-specific elongation occurred in the oligosaccharide that was less susceptible to endo H, indicating that these properties are determined by different conformational constraints.

scholarly journals Robo functions as an attractive cue for glial migration through SYG-1/Neph

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Zhongwei Qu ◽  
Albert Zhang ◽  
Dong Yan
Keyword(s):  
Cell Adhesion ◽  
Caenorhabditis Elegans ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Regulatory Mechanism ◽  
Biological Processes ◽  
Independent Function ◽  
Animal Kingdom ◽  
Wave Complex

As one of the most-studied receptors, Robo plays functions in many biological processes, and its functions highly depend on Slit, the ligand of Robo. Here we uncover a Slit-independent role of Robo in glial migration and show that neurons can release an extracellular fragment of Robo upon cleavage to attract glia during migration in Caenorhabditis elegans. Furthermore, we identified the conserved cell adhesion molecule SYG-1/Neph as a receptor for the cleaved extracellular Robo fragment to mediate glial migration and SYG-1/Neph functions through regulation of the WAVE complex. Our studies reveal a previously unknown Slit-independent function and regulatory mechanism of Robo and show that the cleaved extracellular fragment of Robo can function as a ligand for SYG-1/Neph to guide glial migration. As Robo, the cleaved region of Robo, and SYG-1/Neph are all highly conserved across the animal kingdom, our findings may present a conserved Slit-independent Robo mechanism during brain development.

scholarly journals Missing the sweet spot: one of the two N-glycans on human Gb3/CD77 synthase curtails its activity

2021 ◽  
Author(s):  
Krzysztof Mikolajczyk ◽  
Anna Bereznicka ◽  
Katarzyna Szymczak-Kulus ◽  
Katarzyna Haczkiewicz-Lesniak ◽  
Bozena Szulc ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Subcellular Localization ◽  
Posttranslational Modification ◽  
Regulatory Mechanism ◽  
Specific Activity ◽  
Sweet Spot ◽  
Dual Nature ◽  
Glycosylation Sites ◽  
Opposing Effects

AbstractN-glycosylation is a ubiquitous posttranslational modification that may influence folding, subcellular localization, secretion, solubility and oligomerization of proteins. In this study, we examined the role of N-glycans in the activity of human Gb3/CD77 synthase, which catalyzes the synthesis of glycosphingolipids with terminal Galα1→4Galβ (Gb3 and the P1 antigen) and Galα1→4GalNAcβ disaccharides (the NOR antigen). The human Gb3/CD77 synthase contains two occupied N-glycosylation sites at positions N121 and N203. Intriguingly, we found that while the N-glycan at N203 is essential for activity and correct subcellular localization, the N-glycan at N121 is dispensable and a glycoform without it showed increased enzyme activity. The fully N-glycosylated human Gb3/CD77 synthase and its glycoform missing the N121 glycan correctly localize in the Golgi, whereas a glycoform without the N203 site partially mislocalized in the endoplasmic reticulum. A double mutein missing both N-glycans was inactive and accumulated in the ER. Our results suggest that the decreased specific activity of human Gb3/CD77 synthase glycovariants results from their improper subcellular localization and, to a smaller degree, a decrease in enzyme solubility. Taken together, our findings show that the two N-glycans of human Gb3/CD77 synthase have opposing effects on its properties, revealing a dual nature of N-glycosylation and potentially a novel regulatory mechanism controlling biological activity of proteins.

Binding of hexabrachion (tenascin) to the extracellular matrix and substratum and its effect on cell adhesion

1990 ◽  
Vol 95 (2) ◽  
pp. 263-277
Author(s):  
V.A. Lightner ◽  
H.P. Erickson
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Cell Attachment ◽  
Solid Phase ◽  
Fluid Phase ◽  
Matrix Proteins ◽  
And Migration ◽  
Cover Up ◽  
Plastic Substratum

Hexabrachion is a large glycoprotein of the extracellular matrix (ECM) that is prominent in embryogenesis, wound healing and tumorigenesis. Because of the role of extracellular matrix proteins in the regulation of cell differentiation and migration, the interaction of hexabrachion with cells as well as with other components of the ECM is of great interest. Early reports suggested that hexabrachion does not bind to fibronectin or gelatin but does bind to chondroitin sulfate proteoglycans. However, more recent reports have suggested that hexabrachion binds to fibronectin and inhibits cell adhesion as well as cell migration on fibronectin. We have found no evidence of strong hexabrachion-fibronectin binding on either a solid-phase ELISA assay or in a fluid-phase sedimentation assay in which the reactants were allowed to dissociate. However, hexabrachion sedimentation was accelerated in a gradient containing fibronectin throughout. This demonstrates an association between hexabrachions and fibronectin, but the complex is apparently weak and readily reversible. The solid-phase ELISA also shows no evidence of hexabrachion binding to gelatin, laminin or types I, III, IV or V collagen. Hexabrachion does not support strong cell attachment of the cell lines tested. Moreover, hexabrachion can inhibit cell attachment to fibronectin. We demonstrate here that this inhibition requires the hexabrachion to be able to bind to the plastic substratum. The results suggest that hexabrachion inhibition is via a steric inhibition. When the hexabrachion molecules bind to the plastic, they cover up a significant fraction of the underlying fibronectin molecules. Antibody studies are presented that show that hexabrachion can nonspecifically block access of immunoglobulin G molecules to the underlying matrix. This steric blocking is not unique to hexabrachion.

Sign in / Sign up

Export Citation Format

Share Document