Transglutaminase reactivity of human involucrin.
Human involucrin (hINV) is assembled into cornified structures via formation of transglutaminase (TG)-dependent interprotein epsilon-(gamma-glutamyl)lysine bonds. The hINV sequence includes 150 glutamine residues that could function as potential sites of cross-link formation. The present studies were designed to evaluate the extent to which hINV can function as a TG substrate under optimal conditions and in the absence of other substrates. Incubation of hINV with TG results in formation of 4-5 isopeptide bonds per hINV molecule. When the small amine donor (14)C-putrescine is included in the reaction, 48 Q residues are labled. Isotope distribution and sequence analysis suggests that the (14)C-putrescine-labeled sites are located throughout the protein. Our present results show that many hINV Q residues can be utilized for cross-link formation, and that hINV can be cross-linked at very high cross-link densities. These results suggest that, in vivo, factors other than hINV structure limit the number of residues used for cross-link formation.
Lambert
A
A
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, USA.
Ekambaram
M
M
Robinson
N
N
Eckert
R L
RL
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D019286:Endopeptidase K / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D015603:Keratinocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011498:Protein Precursors / Q000378:metabolism*; D011700:Putrescine / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D011503:Transglutaminases / Q000378:metabolism*; D014357:Trypsin
D003432:Cross-Linking Reagents; D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; C032253:involucrin; D011503:Transglutaminases; D014357:Trypsin; D019286:Endopeptidase K; D011700:Putrescine