Disulfide bonds of GM2 synthase homodimers. Antiparallel orientation of the catalytic domains.
GM2 synthase is a homodimer in which the subunits are joined by lumenal domain disulfide bond(s). To define the disulfide bond pattern of this enzyme, we analyzed a soluble form by chemical fragmentation, enzymatic digestion, and mass spectrometry and a full-length form by site-directed mutagenesis. All Cys residues of the lumenal domain of GM2 synthase are disulfide bonded with Cys(429) and Cys(476) forming a disulfide-bonded pair while Cys(80) and Cys(82) are disulfide bonded in combination with Cys(412) and Cys(529). Partial reduction to produce monomers converted Cys(80) and Cys(82) to free thiols while the Cys(429) to Cys(476) disulfide remained intact. CNBr cleavage at amino acid 330 produced a monomer-sized band under nonreducing conditions which was converted upon reduction to a 40-kDa fragment and a 24-kDa myc-positive fragment. Double mutation of Cys(80) and Cys(82) to Ser produced monomers but not dimers. In summary these results demonstrate that Cys(429) and Cys(476) form an intrasubunit disulfide while the intersubunit disulfides formed by both Cys(80) and Cys(82) with Cys(412) and Cys(529) are responsible for formation of the homodimer. This disulfide bond arrangement results in an antiparallel orientation of the catalytic domains of the GM2 synthase homodimer.
Li
J
J
Department of Molecular, Cellular, and Craniofacial Biology, School of Dentistry, University of Louisville, Louisville, Kentucky 40292, USA.
Yen
T Y
TY
Allende
M L
ML
Joshi
R K
RK
Cai
J
J
Pierce
W M
WM
Jaskiewicz
E
E
Darling
D S
DS
Macher
B A
BA
Young
W W
WW
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D020134:Catalytic Domain; D006224:Cricetinae; D019281:Dimerization; D004220:Disulfides; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry*; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D004220:Disulfides; D017350:N-Acetylgalactosaminyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase