title: Interactions of galactosyltransferase with serum and secretory immunoglobulins and their component chains.
abstract: Assay of the activity of beta-1,4-galactosyltransferase (beta-1,4-GT) revealed that in addition to serum, milk, colostrum, amniotic and cerebrospinal fluids and malignant effusions, this enzyme is present also in tears and saliva. Molecular-sieve chromatography of human colostral whey and serum and subsequent assay of beta-1,4-GT activity have shown that beta-1,4-GT was present as a free enzyme (55 kDa) and associated with components of larger molar mass. The elution pattern did not change when the chromatography was carried out in a buffer devoid of, or enriched with, Mn2+, a cofactor of beta-1,4-GT activity. However, the activity associated with the large molar mass components was absent when the chromatography was carried out in the presence of a chelating agent (EDTA). Analyses of the eluted material by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and by immunodiffusion indicated that the major colostral component in beta-1,4-GT activity-containing fractions was secretory IgA (S-IgA); in addition, the beta-1,4-GT activity was detected in fractions that contained lactoferrin and alpha-lactalbumin. Interactions of beta-1,4-GT with S-IgA and lactoferrin in colostrum were also demonstrated by the detection of radioactivity in precipitin lines obtained by immunoelectrophoresis and autoradiography of the colostral whey after it had been incubated with UDP-[3H]-galactose. Furthermore, radioactively labeled S-IgA and alpha-chain were detected when colostral whey incubated with UDP-[3H]-galactose was analyzed by SDS-PAGE under non-reducing and reducing conditions, respectively. In serum, the beta-1,4-GT-binding components identified in fractions after molecular-sieve chromatography were IgG, IgA, IgM and transferrin. The binding of beta-1,4-GT to immunoglobulins (Ig) was also demonstrated by assaying the beta-1,4-GT activity associated with Sepharose-4B-immobilized Ig of various isotypes and molecular forms, which were incubated with colostral beta-1,4-GT in the presence of Mn2+. Beta-1,4-GT measured by enzyme activity was bound to these Ig in order: polymeric IgA2 > monomeric IgA1 = polymeric IgA1 = secretory IgA = pentameric IgM > IgG. Immobilized component chains, namely alpha, mu and J chains, bound beta-1,4-GT more effectively than native Ig. Incubation of the IgA1 myeloma protein with crude human colostral galactosyltransferase in the presence of UDP[3H]-galactose and Mn2+ resulted in galactosylation of both N- and O-linked carbohydrate side chains.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Medicine, University of Alabama, Birmingham 35294.
authors.forename: M
authors.initials: M
authors.lastname: Tomana
authors.forename: J
authors.initials: J
authors.lastname: Zikan
authors.forename: R
authors.initials: R
authors.lastname: Kulhavy
authors.forename: J C
authors.initials: JC
authors.lastname: Bennett
authors.forename: J
authors.initials: J
authors.lastname: Mestecky
mesh_terms: D000328:Adult; D002850:Chromatography, Gel; D003126:Colostrum / Q000201:enzymology; D005700:Galactosyltransferases / Q000097:blood / Q000276:immunology / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007071:Immunoglobulin A, Secretory / Q000378:metabolism*; D007136:Immunoglobulins / Q000378:metabolism*; D008875:Middle Aged
chemical_list: D006023:Glycoproteins; D007071:Immunoglobulin A, Secretory; D007136:Immunoglobulins; D005700:Galactosyltransferases