Identification of Gln726 in nidogen as the amine acceptor in transglutaminase-catalyzed cross-linking of laminin-nidogen complexes.
The laminin-nidogen complex, the most abundant noncollagenous component of basement membranes, was recently shown to be a specific substrate for tissue transglutaminase (Aeschlimann, D., and Paulsson, M. (1991) J. Biol. Chem. 266, 15308-15317). Saturation experiments to determine the number of amine acceptor site(s) indicated a single reactive Gln residue in nidogen and none in laminin. Murine nidogen was labeled with [3H]putrescine in the tissue transglutaminase-catalyzed reaction, and two major radioactively labeled fragments, T70 and T40, were isolated after limited trypsin digestion. NH2-terminal sequencing showed that T40 is contained in T70 and corresponds to the rodlike structure of nidogen, made up of epidermal growth factor-like repeats. Three radioactively labeled peptides, obtained by extensive trypsin digestion of reduced and alkylated T40, were sequenced. In all a single residue, Gln726, was found to contain label. Sequencing of additional peptides, obtained after further treatment of the largest radioactively labeled peptide with endoproteinase Asp-N, gave the same result. Gln726 is located in an exposed loop between the second and the third EGF-like repeat in nidogen. This site is also conserved in the human sequence.
Aeschlimann
D
D
M. E. Müller Institute for Biomechanics, University of Bern, Switzerland.
Paulsson
M
M
Mann
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine / Q000235:genetics / Q000378:metabolism*; D006168:Guinea Pigs; D007797:Laminin / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010455:Peptides / Q000302:isolation & purification; D011487:Protein Conformation; D011700:Putrescine / Q000378:metabolism; D011503:Transglutaminases / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D003432:Cross-Linking Reagents; D007797:Laminin; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D010455:Peptides; C029967:nidogen; D005973:Glutamine; D011503:Transglutaminases; D014357:Trypsin; D011700:Putrescine