Assay of galactosyltransferase by high-performance liquid chromatography

Aj, Hymes; Mullinax, Franklin

doi:10.1016/0003-2697(84)90389-0

Cited by 14 publications

(8 citation statements)

References 16 publications

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“…(Parekh et at1, 1988), and has been suggested to contnrbute to! the pathology of rheumatoid arthritis, perhaps through a predisposition to self-associate into immunecompleaes (Rademacher et at, .1999) Several other studies; have also indicated an effect of Fab and Fc glycosylation on pharmacologically important parameters (Hymes & Mullinax, 1979;Malaise et at, 1987;Middagh & Litman., 1987).…”

Section: Discussionmentioning

confidence: 96%

Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody

Patel¹,

Parekh²,

Moellering

et al. 1992

Biochemical Journal

127

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A monoclonal IgG-1 was produced by culture of a murine hybridoma (3.8.6) by three different methods, namely culture in ascites, in serum-free media and in serum-supplemented media. IgG-1 was purified to homogeneity (as judged by SDS/PAGE under reducing conditions) from each medium by ion-exchange chromatography and h.p.l.c. Protein A chromatography. Oligosaccharides were released from each IgG-1 preparation by hydrazinolysis and radiolabelled by reduction with alkaline sodium borotritide, and 'profile' analysis of the radiolabelled oligosaccharide alditols was performed by a combination of paper electrophoresis and gel-filtration chromatography. This analysis indicated clear and reproducible differences in the glycosylation patterns of the three IgG-1 preparations. Sequential exoglycosidase analysis of individual oligosaccharides derived from each IgG-1 preparation was used to define these differences. Ascites-derived material differed from serum-free-culture-derived material only with respect to the content of sialic acid. IgG-1 derived from culture in serum-containing media had an intermediate sialic acid content and a lower incidence of outer-arm galactosylation than the other two preparations. These differences in glycosylation could not be induced in any IgG-1 preparation by incubating purified IgG-1 with ascites or culture medium. It is concluded that the glycosylation pattern of a secreted monoclonal IgG is dependent on the culture method employed to obtain it.

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Section: Discussionmentioning

confidence: 96%

Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody

Patel¹,

Parekh²,

Moellering

et al. 1992

Biochemical Journal

127

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“…Carbohydrates have been indicated to be of significant importance in secretion of Ig molecules (1), recognition of polymeric antigens, (2), and creation of IgG-IgG complexes (3). Likewise complement 'fixation, opsonic activity, and the binding to Fc receptors of rabbit IgG antibodies may require carbohydrate moieties (4,5).…”

mentioning

confidence: 99%

Biological significance of carbohydrate chains on monoclonal antibodies.

Nose¹,

Wigzell

1983

Proc. Natl. Acad. Sci. U.S.A.

365

179

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We have prepared monoclonal hapten-specific mouse IgG2b antibodies depleted of asparagine-linked carbohydrate chains by treating' the hybridoma cells with tunicamycin. 'The carbohydrate-deficient antibodies behaved in an identical manner to the normal antibodies with regard to fine antigen-binding reactivity (a Fab fragment feature) and protein A binding capacity [a, feature requiring integrity at the C2H2 and CH3 domaininteraction regions in the constant region of the heavy chain (CH)I. However, they lost 'the ability to activatevcomplement, to bind to Fc.receptors on macrophages, and to induce antibody-dependent cellular cytotoxicity. Furthermore, antigen-antibody. complexes produced from such carbohydrate-deficient antibodies failed to be eliminated rapidly from the circulation. We conclude that removal of carbohydrate chains from IgG molecules may have a profound and highly select impact on the biological activity to these antibodies.Carbohydrates have been indicated to be of significant importance in secretion of Ig molecules (1) Biosynthetic Radiolabeling of Ig and TNP Binding Assay. Hybridoma cells were put in the labeling medium (F10 medium without L-leucine containing 15% fetal calf serum pretreated with TNP-coated SRBC, 4 mM of L-glutamine, 100 pug of streptomycin per ml, 100 units of penicillin-per ml, and 10 mM Hepes) at a cell density of 1 x 106 cells per ml. In some experiments (see Fig. 1 (Cooke, UK). After incubation for 1 hr at 40C, TNP-SRBC or SRBC were washed in GVB2' dissolved in 0.2 ml of 1% NaDodSO4 solution, and transferred to 2 ml of a 2:1 (vol/vol) toluene/Triton-X-100-based scintillation solution. Ig specifically bound to TNP was determined by subtracting the radioactivity bound to SRBC, which was always <10% of that of TNP-SRBC. Furthermore, adding TNP-bovine serum albumin (TNP-albumin) in excess did inhibit >98% of the binding of Ig to TNP-SRBC (data not included). The antibody level of each sample also was measured by hemagglutination (11) of TNP-SRBC.

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“…In nonenzymatic glycoproteins, the inhibition of biological activities caused by deglycosylation is attributed to the reduced binding of the glycoconjugate to membrane or soluble protein receptors (30)(31)(32)(33). The preliminary report that treatment of algal autolysine with a-glucosidase inhibits its cell wall hydrolytic activity (34) may represent another situation in which enzymebound carbohydrate influences catalytic activity, although in this case the nature of the inhibition was not well characterized.…”

Section: Discussionmentioning

confidence: 99%

Partial deglycosylation of chloroplast coupling factor 1 (CF1) prevents the reconstitution of photophosphorylation.

Maione¹,

Jagendorf²

1984

Proc. Natl. Acad. Sci. U.S.A.

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Treatment of spinach chloroplast coupling factor 1 (CF1) with a mixture of glycosidases resulted in the removal of =75% of the carbohydrate associated with the enzyme. The ATPase of CF1 was not activated by this treatment nor were its heat-activated or methanol-dependent ATPase activities inhibited. The deglycosylated enzyme, however, was unable to catalyze photophosphorylation when recoupled with CF,-depleted thylakoids. The glycosidase-treated protein competed with native CF1 for specific binding sites on the depleted membranes and was able to reconstitute proton uptake in uncoupled thylakoids. The inhibition of photophosphorylation could not be overcome with added 6 subunit. We conclude that deglycosylated CF1 was unaffected in its ability to bind to the membrane sector of the chloroplast proton-pumping ATPase (CFO) but was altered in some property essential for photophosphorylation but not ATPase activity.

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Assay of galactosyltransferase by high-performance liquid chromatography

Cited by 14 publications

References 16 publications

Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody

Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody

Biological significance of carbohydrate chains on monoclonal antibodies.

Partial deglycosylation of chloroplast coupling factor 1 (CF1) prevents the reconstitution of photophosphorylation.

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