Glycosaminoglycans modulate fibronectin matrix assembly and are essential for matrix incorporation of tenascin-C

1997 ◽  
Vol 110 (12) ◽  
pp. 1413-1419 ◽  
Author(s):  
C.Y. Chung ◽  
H.P. Erickson
Keyword(s):  
Heparan Sulfate ◽  
Splice Variant ◽  
Cultured Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Matrix Assembly ◽  
Tenascin C ◽  
Sulfate Production ◽  
Fibronectin Matrix ◽  
The Matrix

We have investigated the role of glycosaminoglycans in fibronectin matrix assembly and the incorporation of tenascin-C into matrix fibrils. Chinese hamster ovary cell mutants with a total block in heparan and chondroitin sulfate production failed to assemble a fibronectin matrix, and incorporated no tenascin-C. Another mutant with reduced heparan sulfate produced a normal fibronectin matrix but failed to incorporate tenascin-C. Excess soluble glycosaminoglycans inhibited the binding of tenascin-C to purified fibronectin in ELISA, and completely blocked incorporation into matrix fibrils. Treating cultured cells with xyloside, which interferes with glycosaminoglycan attachment to proteoglycans, also completely blocked their ability to incorporate tenascin-C into matrix fibrils. We conclude that proteoglycans bound to fibronectin fibrils play a major role in binding tenascin-C to these fibrils. We examined more closely the large heparan sulfate proteoglycan, perlecan, and found that it co-localizes with tenascin-C and fibronectin in the matrix. The perlecan binding site in tenascin-C was mapped to the fibronectin type III domains 3–5, but this binding was strongly enhanced for the small splice variant, which is the major form incorporated into the matrix. Apparently when the alternative splice segment is inserted after domain 5 it inhibits perlecan binding. Thus heparan sulfate glycosaminoglycans, and perlecan in particular, may play a role in incorporation of the small splice variant of tenascin-C into fibronectin matrix fibrils.

scholarly journals Characterization of a Chinese Hamster Ovary Cell Line Developed by Retroviral Insertional Mutagenesis That Is Resistant to Sindbis Virus Infection

1999 ◽  
Vol 73 (6) ◽  
pp. 4919-4924 ◽  
Author(s):  
Jia-Tsrong Jan ◽  
Andrew P. Byrnes ◽  
Diane E. Griffin
Keyword(s):  
Heparan Sulfate ◽  
Cell Line ◽  
Virus Replication ◽  
Chinese Hamster Ovary ◽  
Sindbis Virus ◽  
Insertional Mutagenesis ◽  
Cultured Cells ◽  
Chinese Hamster ◽  
Ovary Cell

ABSTRACT The alphavirus Sindbis virus (SV) has a wide host range and infects many types of cultured cells in vitro. The outcome of infection is dependent on the strain of virus used for infection and the properties of the cells infected. To identify cellular determinants of susceptibility to SV infection we mutagenized Chinese hamster ovary (CHO) cells by retroviral insertion with a vector containing the neomycin resistance gene that allowed selection for integration into transcriptionally active genes. Cells were then selected for survival after infection with SV. The most resistant cell line (CHO-18.4m) exhibited delayed virus replication and virus-induced cell death, had a single retroviral insertion, and was defective in SV binding to the cell surface. Further analysis revealed that CHO-18.4m cells were deficient in the expression of the sulfated glycosaminoglycans heparan sulfate and chondroitin sulfate. This further confirms the importance of heparan sulfate as an attachment molecule for SV in vitro and demonstrates the usefulness of this technique for identifying cellular genes that are important for virus replication.

Control of extracellular matrix assembly by syndecan-2 proteoglycan

2000 ◽  
Vol 113 (3) ◽  
pp. 493-506 ◽  
Author(s):  
C.M. Klass ◽  
J.R. Couchman ◽  
A. Woods
Keyword(s):  
Extracellular Matrix ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Metabolic Labeling ◽  
Sulfate Proteoglycan ◽  
S2 Cells ◽  
Matrix Assembly ◽  
The Matrix ◽  
Beta1 Integrin ◽  
Assembly Defect

Extracellular matrix (ECM) deposition and organization is maintained by transmembrane signaling and integrins play major roles. We now show that a second transmembrane component, syndecan-2 heparan sulfate proteoglycan, is pivotal in matrix assembly. Chinese Hamster Ovary (CHO) cells were stably transfected with full length (S2) or truncated syndecan-2 lacking the C-terminal 14 amino acids of the cytoplasmic domain (S2deltaS). No differences in the amount of matrix assembly were noted with S2 cells, but those expressing S2deltaS could not assemble laminin or fibronectin into a fibrillar matrix. The loss of matrix formation was not caused by a failure to synthesize or externalize ECM components as determined by metabolic labeling or due to differences in surface expression of alpha5 or beta1 integrin. The matrix assembly defect was at the cell surface, since S2deltaS cells also lost the ability to rearrange laminin or fibronectin substrates into fibrils and to bind exogenous fibronectin. Transfection of activated alphaIIbalphaLdeltabeta3 integrin into alpha(5)-deficient CHO B2 cells resulted in reestablishment of the previously lost fibronectin matrix. However, cotransfection of this cell line with S2deltaS could override the presence of activated integrins. These results suggest a regulatory role for syndecan-2 in matrix assembly, along with previously suggested roles for activated integrins.

scholarly journals An Amino Acid Substitution in the Coding Region of the E2 Glycoprotein Adapts Ross River Virus To Utilize Heparan Sulfate as an Attachment Moiety

2001 ◽  
Vol 75 (14) ◽  
pp. 6303-6309 ◽  
Author(s):  
Marintha L. Heil ◽  
Alison Albee ◽  
James H. Strauss ◽  
Richard J. Kuhn
Keyword(s):  
Amino Acid ◽  
Heparan Sulfate ◽  
Amino Acid Substitution ◽  
Cultured Cells ◽  
Wild Type Virus ◽  
Ross River Virus ◽  
Wild Type ◽  
Coding Region ◽  
Sulfate Production ◽  
E2 Glycoprotein

ABSTRACT Passage of Ross River virus strain NB5092 in avian cells has been previously shown to select for virus variants that have enhanced replication in these cells. Sequencing of these variants identified two independent sites that might be responsible for the phenotype. We now demonstrate, using a molecular cDNA clone of the wild-type T48 strain, that an amino acid substitution at residue 218 in the E2 glycoprotein can account for the phenotype. Substitutions that replaced the wild-type asparagine with basic residues had enhanced replication in avian cells while acidic or neutral residues had little or no observable effect. Ross River virus mutants that had increased replication in avian cells also grew better in BHK cells than the wild-type virus, whereas the remaining mutants were unaffected in growth. Replication in both BHK and avian cells of Ross River virus mutants N218K and N218R was inhibited by the presence of heparin or by the pretreatment of the cells with heparinase. Binding of the mutants, but not of the wild type, to a heparin-Sepharose column produced binding comparable to that of Sindbis virus, which has previously been shown to bind heparin. Replication of these mutants was also adversely affected when they were grown in a CHO cell line that was deficient in heparan sulfate production. These results demonstrate that amino acid 218 of the E2 glycoprotein can be modified to create an heparan sulfate binding site and this modification expands the host range of Ross River virus in cultured cells to cells of avian origin.

scholarly journals CD98hc (SLC3A2) participates in fibronectin matrix assembly by mediating integrin signaling

2007 ◽  
Vol 178 (4) ◽  
pp. 701-711 ◽  
Author(s):  
Chloé C. Féral ◽  
Andries Zijlstra ◽  
Eugene Tkachenko ◽  
Gerald Prager ◽  
Margaret L. Gardel ◽  
...  
Keyword(s):  
Wound Healing ◽  
Membrane Protein ◽  
Small Gtpase ◽  
Integrin Signaling ◽  
Vertebrate Development ◽  
Matrix Assembly ◽  
Fibronectin Matrix ◽  
The Matrix

Integrin-dependent assembly of the fibronectin (Fn) matrix plays a central role in vertebrate development. We identify CD98hc, a membrane protein, as an important component of the matrix assembly machinery both in vitro and in vivo. CD98hc was not required for biosynthesis of cellular Fn or the maintenance of the repertoire or affinity of cellular Fn binding integrins, which are important contributors to Fn assembly. Instead, CD98hc was involved in the cell's ability to exert force on the matrix and did so by dint of its capacity to interact with integrins to support downstream signals that lead to activation of RhoA small GTPase. Thus, we identify CD98hc as a membrane protein that enables matrix assembly and establish that it functions by interacting with integrins to support RhoA-driven contractility. CD98hc expression can vary widely; our data show that these variations in CD98hc expression can control the capacity of cells to assemble an Fn matrix, a process important in development, wound healing, and tumorigenesis.

Role of the carboxyl-terminal Fib2 domain in fibronectin matrix assembly

1995 ◽  
Vol 108 (3) ◽  
pp. 907-915 ◽  
Author(s):  
K. Ichihara-Tanaka ◽  
K. Titani ◽  
K. Sekiguchi
Keyword(s):  
Residual Activity ◽  
Significant Activity ◽  
Matrix Assembly ◽  
En Bloc ◽  
Proteolytic Fragment ◽  
Cultured Fibroblasts ◽  
Fibronectin Matrix ◽  
The Matrix ◽  
Assembly Activity

A truncated form of fibronectin consisting of the N-terminal 70 kDa and C-terminal 37 kDa regions, designated r70F2, retained the ability to assemble into the extracellular matrix when expressed in cultured fibroblasts (Ichihara-Tanaka et al. (1992) FEBS Lett. 299, 155–158). To elucidate the role of the C-terminal 37 kDa region in fibronectin matrix assembly, we expressed a panel of mutant forms of r70F2 with various deletions and amino acid substitutions in mouse L cells. Although substitution of Ser for two Cys residues in the C-terminal dimerforming segment led to a marked reduction in the matrix assembly activity of r70F2, the resulting monomeric r70F2 still retained a low, but significant activity to assemble into the matrix. Neither the N-terminal 70 kDa nor the C-terminal 37 kDa regions, when expressed as monomeric forms, exhibited any residual activity, suggesting that the core domain of the 37 kDa region consisting of III15 and I10 through I12 modules, termed Fib2 domain, is actively involved in the matrix assembly of r70F2. In support of the role of Fib2 domain, the proteolytic fragment derived from the 37 kDa region inhibited the assembly of r70F2. Furthermore, en bloc deletion of the Fib2 domain or deletion of the I10 through I12 modules from r70F2 resulted in a marked decrease of the matrix assembly activity.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Cell Surface Heparan Sulfate Promotes Replication of Toxoplasma gondii

2005 ◽  
Vol 73 (9) ◽  
pp. 5395-5401 ◽  
Author(s):  
Joseph R. Bishop ◽  
Brett E. Crawford ◽  
Jeffrey D. Esko
Keyword(s):  
Toxoplasma Gondii ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Cell Types ◽  
Ovary Cell ◽  
Toxoplasma Infection ◽  
Mammary Tumor Cells

ABSTRACT Previous work suggests that cell surface heparan sulfate acts as a receptor for the Apicomplexan parasite Toxoplasma gondii. Using Chinese hamster ovary cell mutants defective in heparan sulfate biosynthesis, we show that heparan sulfate is necessary and sufficient for infectivity. Further, we demonstrate that the parasite requires N sulfation of heparan sulfate initiated by N-deacetylase/N-sulfotransferase-1, but 2-O sulfation and 6-O sulfation appear to be dispensable. In order to study the role of heparan sulfate in other cell types, we created a conditional allele for N-deacetylase/N-sulfotransferase-1 by using Cre-loxP technology. Mammary tumor cells lacking N-deacetylase/N-sulfotransferase-1 exhibited reduced toxoplasma infectivity like Chinese hamster ovary cell mutants. Surprisingly, heparin, chemically modified heparinoids, and monoclonal antibodies to heparan sulfate had no effect on toxoplasma infection. T. gondii attachment and invasion were unchanged in N-deacetylase/N-sulfotransferase-1-inactivated cells as well, but replication was reduced. Thus, heparan sulfate does not appear to function as a receptor for T. gondii but instead facilitates parasite replication postinvasion.

Regulation of fibronectin matrix assembly by tenascin-C

2008 ◽  
Vol 27 ◽  
pp. 14
Author(s):  
Wing S. To ◽  
Hideaki Nagase ◽  
Kim S. Midwood
Keyword(s):  
Matrix Assembly ◽  
Tenascin C ◽  
Fibronectin Matrix
Sign in / Sign up

Export Citation Format

Share Document