Reactivity of fish and microbial transglutaminases on glutaminyl sites of peptides derived from threadfin bream myosin.
Fish liver transglutaminase (FTG), a Ca(2+)-dependent enzyme, exhibits different characteristics from the Ca(2+)-independent microbial transglutaminase (MTG), leading to potential differences in their substrate specificity and reactivity. The ability of these enzymes to catalyze isopeptide bond formation by incorporating 5-(biotinamido)pentylamine (BPNH2) into peptides derived by tryptic digestion of threadfin bream (TB)-myosin was investigated to identify reaction sites and substrate specificity using a peptidomic strategy. BPNH2 was incorporated into TB-myosin peptides to a greater extent by MTG than FTG. Peptides derived from TB-myosin heavy chain (MHC) shared highest similarity to amberjack-MHC on the basis of a Mascot database search. Amino acid sequences and modification sites of BPNH2-tagged peptides were identified by tandem mass spectrometry based on the amberjack-MHC sequence. The BPNH2 modification sites catalyzed by both TGases were at the myosin rod. Most of the BPNH2 peptides contained charged amino acids (E, R, K) at the glutaminylamide site of reactive glutamine (Q*). The alpha-acrylamide site of Q* contained E, F, or L on peptides catalyzed by both enzymes, I, Q, or A on peptides catalyzed only by FTG, and V on a peptide catalyzed only by MTG. These results demonstrate the different structural requirements for glutaminyl substrates between these two enzymes.
Hemung
Bung-Orn
BO
School of Food Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Li-Chan
Eunice C Y
EC
Yongsawatdigul
Jirawat
J
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D005399:Fishes; D005973:Glutamine / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D005973:Glutamine; D011503:Transglutaminases; D014357:Trypsin; D009218:Myosins; D002118:Calcium