Disulphide linkage in mouse ST6Gal-I: determination of linkage positions and mutant analysis.
All cloned sialyltransferases from vertebrates are classified into four subfamilies and are characterized as having type II transmembrane topology. The catalytic domain has highly conserved motifs known as sialylmotifs. Besides sialylmotifs, each family has several unique conserved cysteine (Cys) residues mainly in the catalytic domain. The number and loci of conserved amino acids, however, differ with each subfamily, suggesting that the conserved Cys-residues and/or disulphide linkages they make may contribute to linkage specificity. Using Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF)-mass spectrometry, the present study performed disulphide linkage analysis on soluble mouse ST6Gal-I, which has six Cys-residues. Results confirmed that there were no free Cys-residues, and all six residues contributed to disulphide linkage formation, C(139)-C(403), C(181)-C(332) and C(350)-C(361). Study of single amino acid-substituted mutants revealed that the disulphide linkage C(181)-C(332) was necessary for molecular expression of the enzyme, and that the disulphide linkage C(350)-C(361) was necessary for enzyme activity. The remaining disulphide linkage C(139)-C(403) was not necessary for enzyme expression or for activity, including substrate specificity. Crystallographic study of pig ST3Gal I has recently been reported. Interestingly, the loci of disulphide linkages in ST6Gal-I differ from those in ST3Gal I, suggesting that the linkage specificity of sialyltransferase may results from significant structural differences, including the loci of disulphide linkages.
Institute of Glycoscience, Tokai University, 4-1-1 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan.
Yuichi
Y
Hirano
Takehiro
T
Suzuki
Takumi
T
Matsumoto
Yoshimi
Y
Ishihara
Yoshie
Y
Takaki
Mari
M
Kono
Naoshi
N
Dohmae
Shuichi
S
Tsuji
D000818:Animals; D019556:COS Cells; D002233:Carbocysteine / Q000737:chemistry / Q000378:metabolism; D002522:Chlorocebus aethiops; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012799:Sialyltransferases / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D004220:Disulfides; D002233:Carbocysteine; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase