TY - JOUR
T1 - Heparan sulfate is necessary for the early formation of nascent fibronectin and collagen I fibrils at matrix assembly sites
AU - Hill, Katherine E.
AU - Lovett, Benjamin M.
AU - Schwarzbauer, Jean E.
N1 - Funding Information:
This research was funded by grants (to J. E. S.) from the National Institutes of Health NIAMS R01 AR073236 and the Sud Cook'39 Fund for the Prevention of Addiction to Alcohol and other Mind-altering Drugs. K. E. H. and B. M. L. were supported by a NIH T32 GM007388 predoctoral training grant to the Department of Molecular Biology at Princeton University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
Funding and additional information—This research was funded by grants (to J. E. S.) from the National Institutes of Health NIAMS R01 AR073236 and the Sud Cook ’39 Fund for the Prevention of Addiction to Alcohol and other Mind-altering Drugs. K. E. H. and B. M. L. were supported by a NIH T32 GM007388 predoctoral training grant to the Department of Molecular Biology at Princeton University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
PY - 2022/1/1
Y1 - 2022/1/1
N2 - Fibronectin (FN), an essential component of the extracellular matrix (ECM), is assembled via a cell-mediated process in which integrin receptors bind secreted FN and mediate its polymerization into fibrils that extend between cells, ultimately forming an insoluble matrix. Our previous work using mutant Chinese hamster ovary (CHO) cells identified the glycosaminoglycan heparan sulfate (HS) and its binding to FN as essential for the formation of insoluble FN fibrils. In this study, we investigated the contributions of HS at an early stage of the assembly process using knockdown of exostosin-1 (EXT1), one of the glycosyltransferases required for HS chain synthesis. NIH 3T3 fibroblasts with decreased EXT1 expression exhibited a significant reduction in both FN and type I collagen in the insoluble matrix. We show that FN fibril formation is initiated at matrix assembly sites, and while these sites were formed by cells with EXT1 knockdown, their growth was stunted compared with wild-type cells. The most severe defect observed was in the polymerization of nascent FN fibrils, which was reduced 2.5-fold upon EXT1 knockdown. This defect was rescued by the addition of exogenous soluble heparin chains long enough to simultaneously bind multiple FN molecules. The activity of soluble heparin in this process indicates that nascent fibril formation depends on HS more so than on the protein component of a specific HS proteoglycan. Together, our results suggest that heparin or HS is necessary for concentrating and localizing FN molecules at sites of early fibril assembly.
AB - Fibronectin (FN), an essential component of the extracellular matrix (ECM), is assembled via a cell-mediated process in which integrin receptors bind secreted FN and mediate its polymerization into fibrils that extend between cells, ultimately forming an insoluble matrix. Our previous work using mutant Chinese hamster ovary (CHO) cells identified the glycosaminoglycan heparan sulfate (HS) and its binding to FN as essential for the formation of insoluble FN fibrils. In this study, we investigated the contributions of HS at an early stage of the assembly process using knockdown of exostosin-1 (EXT1), one of the glycosyltransferases required for HS chain synthesis. NIH 3T3 fibroblasts with decreased EXT1 expression exhibited a significant reduction in both FN and type I collagen in the insoluble matrix. We show that FN fibril formation is initiated at matrix assembly sites, and while these sites were formed by cells with EXT1 knockdown, their growth was stunted compared with wild-type cells. The most severe defect observed was in the polymerization of nascent FN fibrils, which was reduced 2.5-fold upon EXT1 knockdown. This defect was rescued by the addition of exogenous soluble heparin chains long enough to simultaneously bind multiple FN molecules. The activity of soluble heparin in this process indicates that nascent fibril formation depends on HS more so than on the protein component of a specific HS proteoglycan. Together, our results suggest that heparin or HS is necessary for concentrating and localizing FN molecules at sites of early fibril assembly.
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U2 - 10.1016/j.jbc.2021.101479
DO - 10.1016/j.jbc.2021.101479
M3 - Article
C2 - 34890641
AN - SCOPUS:85122148722
SN - 0021-9258
VL - 298
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 1
M1 - 101479
ER -