Radiation inactivation of human intestinal mucin: determination of the size of the functional antigenic unit.
Previously we have shown that the major antigenic determinant of human intestinal mucin is associated with its glycopeptide monomers and not the 118 kDa 'link' component. In the present study, the size and nature of the functional unit containing the antigenic determinant has been assessed by radiation inactivation and immunological assays. Increasing doses of radiation led to a monoexponential decay in antigenic reactivity due to a progressive loss of antigenic determinants. From three independent mucin preparations, a value of 78500 +/- 7000 was determined for the Mr of the functional antigenic unit. Prolonged pronase digestion of native mucin released large degraded glycopeptide monomers containing all the mucin carbohydrate, and low molecular weight peptides. The antigenicity of the glycopeptides decreased with digestion but could not be recovered in the peptide fractions, suggesting that determinants were released and destroyed by the enzyme. Treatment of native mucin with trifluoromethanesulphonic acid caused a major loss of carbohydrate (approx. 70%), but the protein component was unchanged in amino acid profile and remained antigenic. Subsequent thiol reduction, however, abolished the antigenicity of the deglycosylated mucin. We conclude that antigenicity is associated with a non-glycosylated segment of the peptide backbone of the glycopeptides and that a large functional unit of Mr 78500 which is stabilized by disulphide bonds is important for full antigenic activity.
Mantle
M
M
Potier
M
M
Forstner
G G
GG
Forstner
J F
JF
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002850:Chromatography, Gel; D003037:Cobalt Radioisotopes; D000939:Epitopes / Q000528:radiation effects*; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D007422:Intestines / Q000032:analysis*; D046911:Macromolecular Substances; D008698:Mesylates / Q000494:pharmacology; D008970:Molecular Weight; D009077:Mucins / Q000528:radiation effects*; D011402:Pronase / Q000378:metabolism; D011863:Radioimmunoassay
D000596:Amino Acids; D002241:Carbohydrates; D003037:Cobalt Radioisotopes; D000939:Epitopes; D006023:Glycoproteins; D046911:Macromolecular Substances; D008698:Mesylates; D009077:Mucins; D011402:Pronase; C012077:trifluoromethanesulfonic acid