Cellular Adhesion Responses to the Heparin-binding (HepII) Domain of Fibronectin Require Heparan Sulfate with Specific Properties

Yashithra Mahalingam,John R Couchman,John T Gallagher

doi:10.1074/jbc.m604938200

Abstract

Cell surface heparan sulfate (HS) proteoglycans are required in development and postnatal repair. Important classes of ligands for HS include growth factors and extracellular matrix macromolecules. For example, the focal adhesion component syndecan-4 interacts with the III(12-14) region of fibronectin (HepII domain) through its HS chains. The fine structure of HS is critical to growth factor responses, and whether this extends to matrix ligands is unknown but is suggested from in vitro experiments. Cell attachment to HepII showed that heparin oligosaccharides of >or=14 sugar residues were required for optimal inhibition. The presence of N-sulfated glucosamine in the HS was essential, whereas 2-O-sulfation of uronic acid or 6-O-sulfation of glucosamine had marginal effects. In the more complex response of focal adhesion formation through syndecan-4, N-sulfates were again required and also glucosamine 6-O-sulfate. The significance of polymer N-sulfation and sulfated domains in HS was confirmed by studies with mutant Chinese hamster ovary cells where heparan sulfation was compromised. Finally, focal adhesion formation was absent in fibroblasts synthesizing short HS chains resulting from a gene trap mutation in one of the two major glucosaminoglycan polymerases (EXT1). Several separate, specific properties of cell surface HS are therefore required in cell adhesion responses to the fibronectin HepII domain.

Highlights

The percentage was calculated by analyzing a minimum of 50 treated or nontreated cells for focal adhesions
Cells containing more than five distinct focal adhesions were deemed positive
When cells pre-spread on the 110-kDa domain were incubated with the HepII domain in the presence of desulfated heparin 14-mers, only cells that had the addition of either CompDes or DNS and to a lesser extent DNRAc were able to bundle their stress fibers and form focal adhesions

Summary

EXPERIMENTAL PROCEDURES

Materials and Antibodies—For cell adhesion assays, heparin (Sigma), heparan sulfate (Seikaguku, Tokyo), chondroitin sulfate C (Seikaguku), and variously desulfated heparin and heparan sulfate oligosaccharides (see below for production methods) were used as competitors. The polyclonal chicken anti-syndecan-4 antibody (Harlan Sera-Lab, UK) was raised against 20 amino acids of the N-terminal human syndecan-4 ectodomain sequence, and antibodies were affinity-purified from plasma using standard procedures This antibody together with Alexa Fluor 488-conjugated goat anti-chicken IgY (Invitrogen) was used for FACS analysis. Cell Culture—Rat embryo fibroblasts [29] were cultured in ␣-minimum Eagle’s medium (Cambrex, Wokingham, UK) containing 5% fetal calf serum (Biowest, Ringmer, UK) and 1% glutamine (Invitrogen). Flow Cytometry Analyses—Cells were detached from flasks with Hanks’-based, enzyme-free, cell dissociation buffer (Invitrogen), washed, resuspended in 1 ϫ 106 cells/ml of FACS buffer (PBS containing 2 mM EDTA and 0.5% BSA), and incubated with 14 ␮g/ml chicken anti-syndecan-4 polyclonal antibody for 20 min at 4 °C. After washing with FACS buffer, the cells were stained with secondary antibody Alexa Fluor 488conjugated goat anti-chicken IgY (1:1000). The cells were analyzed on FACSCalibur, and the CellQuest software (both from BD Biosciences) was used for acquisition and analysis of the data

RESULTS

Chondroitin sulfate C

DISCUSSION