Promotion of selective cell attachment by the RGD sequence in dentine matrix protein 1.
Dentine matrix protein 1 (DMP1) is an important component of the non-collagenous extracellular matrix of developing teeth and bones. Functions of DMP1 other than a putative role in the initiation of mineralization are largely unknown. A first report on the DNA and deduced amino acid sequence showed that DMP1 has a single Arg-Gly-Asp (RGD) sequence. Here, whether the RGD sequence functions as a cell-attachment domain was tested. Using site-directed mutagenesis, two mutant recombinant DMP1 proteins with specific alterations at the RGD site were created. In the first mutant protein the RGD sequence was altered to a RGE (RGE) sequence; in the second the RGD domain was deleted (DEL). Mutated proteins were confirmed to be DMP1 by partial protein sequencing and dot-blot analysis with an anti-DMP1 antibody. Attachment of RPC-C2A (dental pulp cells), MC3T3-E1 (calvarial cells) or CHO (Chinese hamster ovary cells) to non-tissue-culture plastic coated with either DMP1, RGE or DEL proteins was compared. Bovine serum albumin and fibronectin served as negative and positive controls, respectively. The RGD-containing native DMP1 protein effectively allowed cell attachment and spreading. The RGE and DEL proteins with the altered and deleted RGD sites were significantly less effective in promoting cell attachment than the recombinant DMP1. Both RPC-C2A pulp cells and MC3T3-E1 cells showed similar reductions in attachment to mutated proteins. Treatment of RPC-C2A cells with a RGD-containing peptide prior to plating on DMP1-coated chambers abolished DMP1-mediated cell attachment. In contrast to RPC-C2A and MC3T3-E1cells, CHO cells, which normally do not express DMP1, failed to attach to DMP1. These data demonstrate that DMP1 promotes cell attachment through the RGD domain and that the attachment is cell- and tissue-specific. A basis for these observations is proposed using computer-generated models of the polypeptides within the DMP1 protein containing the RGD, RGE or DEL sequences.
Kulkarni
G V
GV
Room 455D, Faculty of Dentistry, University of Toronto, 124 Edward Street, Toronto, Canada.
Chen
B
B
Malone
J P
JP
Narayanan
A S
AS
George
A
A
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000032:analysis; D001224:Aspartic Acid / Q000032:analysis; D016466:CHO Cells; D002417:Cattle; D002448:Cell Adhesion / Q000502:physiology*; D006224:Cricetinae; D003782:Dental Pulp / Q000166:cytology*; D003804:Dentin / Q000737:chemistry* / Q000502:physiology; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000502:physiology*; D005998:Glycine / Q000032:analysis; D015151:Immunoblotting; D051379:Mice; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000737:chemistry* / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D020539:Sequence Analysis, Protein
C513308:Dmp1 protein, mouse; D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D001120:Arginine; D005998:Glycine