4.2 Article

Disulphide linkage in mouse ST6Gal-I: determination of linkage positions and mutant analysis

Journal

JOURNAL OF BIOCHEMISTRY
Volume 151, Issue 2, Pages 197-203

Publisher

OXFORD UNIV PRESS
DOI: 10.1093/jb/mvr133

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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.

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