Identification of the epsilon-(gamma-glutamyl)lysine cross-linking sites in alpha-lactalbumin polymerized by mammalian and microbial transglutaminases.
To investigate the site specificity of two transglutaminases (TGases), that is, the enzymes from guinea pig liver (GTGase) and Streptoverticillium (MTGase), the acyl acceptor and donor sites in alpha-lactalbumin were determined. Alpha-lactalbumin was cross-linked in the presence of dithiothreitol by GTGase and MTGase for 15 and 30 min, respectively. Cross-linked alpha-lactalbumins by GTGase and MTGase were digested with lysylendopeptidase followed by the separation of the resulting peptides using reverse-phase HPLC. By the sequence analysis of the peptide fragments containing two N termini, which indicates the presence of cross-linked peptide, the lysine residues targeted by TGases were identified as follows: for GTGase, Lys16, Lys93, and Lys122; for MTGase, Lys5. These peptide fragments were further digested by V8 protease. Separation and sequence analyses of the resultant peptides were performed to identify glutamine residue involved in cross-linking. It was found that Gln54 was cross-linked to lysine residues by GTGase and MTGase in common. It is suggested that the difference in the numbers of lysine residues targeted by GTGase and MTGase may be responsible for the difference in the polymerization process of alpha-lactalbumin between GTGase- and MTGase-catalyzed systems.
Lee
Deuk-Sik
DS
Department of Tourism and Foodservice Industry, Donghae University, Jiheungdong, Donghae-shi, Kangwondo 240-713, Korea.
Matsumoto
Shinya
S
Matsumura
Yasuki
Y
Mori
Tomohiko
T
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D005973:Glutamine / Q000378:metabolism*; D006168:Guinea Pigs; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism; D013306:Streptomycetaceae / Q000201:enzymology; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D005973:Glutamine; D007768:Lactalbumin; D011503:Transglutaminases; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase; D008239:Lysine