Abstract
Microfibril-associated glycoprotein (MAGP)-1 and MAGP-2 are small structurally related glycoproteins that are specifically associated with fibrillin-containing microfibrils. MAGP-2, unlike MAGP-1, contains an RGD motif with potential for integrin binding. To determine if the RGD sequence is active, a series of cell binding assays was performed. MAGP-2 was shown to promote the attachment and spreading of bovine nuchal ligament fibroblasts when coated onto plastic wells in molar quantities similar to those of fibronectin. In contrast, approximately 10-fold more MAGP-1 was required to support comparable levels of cell adhesion. The fibroblast binding to MAGP-2 was completely inhibited if the peptide GRGDSP or the MAGP-2-specific peptide GVSGQRGDDVTTVTSET was added to the reaction medium at a 10 microM final concentration. The control peptide GRGESP had no effect on the interaction. These findings indicate that the cell interaction with MAGP-2 is an RGD-mediated event. A monoclonal antibody to human alphaVbeta3 integrin (LM609) almost completely blocked cell attachment to MAGP-2 when added to the medium at 0.5 microgram/ml, whereas two monoclonal antibodies specific for the human beta1 integrin subunit, 4B4 (blocking) and QE2.E5 (activating), had no effect even at 10 microgram/ml. Fetal bovine aortic smooth muscle cells, ear cartilage chondrocytes, and arterial endothelial cells and human skin fibroblasts and osteoblasts were also observed to adhere strongly to MAGP-2. In addition, each cell type was able to spread on MAGP-2 substrate, with the exception of the endothelial cells, which remained spherical after 2 h of incubation. The binding of each cell type was blocked when the anti-alphaVbeta3 integrin antibody was included in the assay, indicating that alphaVbeta3 integrin is the major receptor for MAGP-2 on several cell types. Thus, MAGP-2 may mediate interactions between fibrillin-containing microfibrils and cell surfaces during the development of a variety of tissues.
Highlights
Microfibril-associated glycoprotein (MAGP)-1 and MAGP-2 are small structurally related glycoproteins that are associated with fibrillin-containing microfibrils
Evidence indicates that the N-terminal region of MAGP-1 contains binding sequences for tropoelastin and type VI collagen, and it is possible that the glycoprotein functions on the surface of the microfibrils, as an elastin-binding protein during elastinogenesis and as an anchoring protein mediating the interaction of fibrillin-containing microfibrils and type VI collagen microfibrils [25,26,27]
MAGP-2 Promotes the Adherence and Spreading of a Range of Cell Types from Fetal Bovine Tissues—Using a solid-phase cell attachment assay, MAGP-2 was shown to promote the adherence of nuchal ligament fibroblasts when microtiter wells were coated with subpicomole quantities of the protein (Fig. 1A)
Summary
Materials—MAGP-1 and MAGP-2 were prepared from fetal bovine nuchal ligament as described previously [21]. Other peptides and bovine fibronectin were purchased from Life Technologies, Inc. Antihuman ␣V3 integrin monoclonal antibody LM609 [30] was purchased from Chemicon International, Inc. Anti-human 1 integrin monoclonal antibodies 4B4 [31] and QE2.E5 [32] were obtained from Beckman Coulter Inc. and Dr R. Human skin fibroblasts obtained from normal adult subjects have been described previously [35]. Grown from trabecular bone explants of normal adults, were a kind gift from Dr D. Human cell lines that were used in the binding assay included MO7e (megakaryoblastic) [36], NALM-6 (null lymphoblastoid) [37], BALM-1 (B lymphocytic) [38], and T47D (breast carcinoma) (American Type Culture Collection). Lowed by incubation in the wells at 37 °C for 2 h
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