Setty S, Kim Y, Fields G B, Clegg D O, Wayner E A, Tsilibary E C
Department of Laboratory Medicine and Pathology, School of Medicine, University of Minnesota, Minneapolis, Minnesota 55455, USA.
J Biol Chem. 1998 May 15;273(20):12244-9. doi: 10.1074/jbc.273.20.12244.
Type IV collagen (COL-IV) interacts with a variety of cell types. We present evidence that human mesangial cells (HMC) bind directly to COL-IV, its major triple helical domain, and the main non-collagenous, NC1 domain. A synthetic peptide, HEP-III, and its triple helical counterpart (THP-III), previously reported to be a heparin-binding domain, also promoted approximately 15% adhesion of HMC. HMC bound to solid-phase-immobilized, intact COL-IV (approximately 75%), isolated NC1 domain (approximately 15%), and a pepsin-derived triple helical fragment,which lacks Hep-III (approximately 65%). We further examined inhibition of HMC adhesion to COL-IV and its domains by using anti-integrin antibodies. Blocking monoclonal antibodies against the alpha2 integrin resulted in 70% inhibition of adhesion to COL-IV and 80% inhibition to HEP-III. Moderate inhibition was observed on the NC1 and triple helical fragments. Anti-alpha1 antibodies inhibited the binding of HMC to COL-IV, the NC1, and triple helical domains, but not to peptide HEP-III. Anti-beta1 antibodies inhibited almost completely (>95%) the adhesion to COL-IV, the NC1, and triple helical fragments; inhibition on HEP-III was approximately 30%. Affinity chromatography studies with solid-phase HEP-III and mesangial cell lysate also demonstrated the presence of integrin alpha2 beta1 along with alpha3 beta1. We conclude that alpha2 beta1 and alpha1 beta1 integrins mediate HMC adhesion to COL-IV. Peptide HEP-III is a major, specific site for alpha2 integrin-mediated binding of mesangial cells to COL-IV. Both the alpha1 beta1 and alpha2 beta1 integrins interact with the NC1 and triple helical fragments of COL-IV. Therefore, we demonstrate that several sites for integrin-mediated interactions exist on several collagenous and non-collagenous domains of COL-IV.
IV型胶原（COL-IV）与多种细胞类型相互作用。我们提供的证据表明，人肾小球系膜细胞（HMC）可直接结合COL-IV、其主要的三螺旋结构域以及主要的非胶原NC1结构域。一种合成肽HEP-III及其三螺旋对应物（THP-III），先前报道为肝素结合结构域，也促进了约15%的HMC黏附。HMC与固相固定的完整COL-IV（约75%）、分离的NC1结构域（约15%）以及缺乏HEP-III的胃蛋白酶衍生三螺旋片段（约65%）结合。我们进一步使用抗整合素抗体研究了对HMC黏附于COL-IV及其结构域的抑制作用。针对α2整合素的阻断单克隆抗体导致对COL-IV黏附的70%抑制以及对HEP-III的80%抑制。在NC1和三螺旋片段上观察到中度抑制。抗α1抗体抑制了HMC与COL-IV、NC1和三螺旋结构域的结合，但不抑制与肽HEP-III的结合。抗β1抗体几乎完全抑制（>95%）了对COL-IV、NC1和三螺旋片段的黏附；对HEP-III的抑制约为30%。用固相HEP-III和系膜细胞裂解物进行的亲和层析研究还证明了整合素α2β1以及α3β1的存在。我们得出结论，α2β1和α1β1整合素介导HMC对COL-IV的黏附。肽HEP-III是α2整合素介导系膜细胞与COL-IV结合的主要特异性位点。α1β1和α2β1整合素均与COL-IV的NC1和三螺旋片段相互作用。因此，我们证明在COL-IV的几个胶原和非胶原结构域上存在多个整合素介导相互作用的位点。
