Transglutaminase from rat coagulating gland secretion. Post-translational modifications and activation by phosphatidic acids.
Structural and biochemical characteristics of transglutaminase purified by a rapid chromatographic procedure from the rat coagulating gland (anterior prostate) secretion are reported. Fast atom bombardment mapping and automated Edman degradation experiments allowed us to verify that at least 85% of the entire transglutaminase amino acid sequence is identical to that derived from the cDNA of the major androgen-dependent rat prostate protein called DP1. The enzyme was found NH2 terminally blocked and largely post-translationally modified, since the presence of N-linked oligosaccharides, as well as of complex lipidic structures, was observed. Mass spectral analysis showed that Asn-408 and -488 are the glycosylated sites, the N-linked structures identified belonging to both high-mannose and complex type glycans. The presence of myo-inositol, of glycerol bound fatty acids, and the high content of mannose residues, are in agreement with previous observations suggesting that a lipid anchor is bound to coagulating gland secretion transglutaminase. Furthermore, two tightly bound calcium ions per molecule of enzyme were detected. Finally, a strong stimulation of the enzyme activity in vitro by both SDS and a variety of phosphatidic acids was observed. The reported structural and functional peculiarities should definitively lead to consider the prostate enzyme as a new member (type IV) of the transglutaminase family.
Esposito
C
C
Department of Biochemistry and Biophysics, 2nd University of Naples, 80138 Naples, Italy.
Pucci
P
P
Amoresano
A
A
Marino
G
G
Cozzolino
A
A
Porta
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000032:analysis; D002240:Carbohydrate Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003545:Cysteine; D004789:Enzyme Activation; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006168:Guinea Pigs; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010712:Phosphatidic Acids / Q000494:pharmacology*; D011467:Prostate / Q000201:enzymology*; D011499:Protein Processing, Post-Translational; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D012741:Sexual Maturation; D016339:Spectrometry, Mass, Fast Atom Bombardment; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*
D002413:Cations, Divalent; D006023:Glycoproteins; D009844:Oligosaccharides; D010446:Peptide Fragments; D010712:Phosphatidic Acids; D011503:Transglutaminases; D003545:Cysteine; D002118:Calcium