Characterization and localization of the putative 'link' component in rat small-intestinal mucin.
Rat intestinal mucin is polymerized by a putative 'link' component of Mr 118,000 that can be released from the native mucin by thiol reduction [Fahim, Forstner & Forstner (1983) Biochem. J. 209, 117-124]. To confirm that this component is an integral part of the mucin and independent of the mucin purification technique, rat mucin was purified in the present study by three independent techniques. In all cases, the 118,000-Mr component was released after reduction. The 118 kDa band was electroeluted from SDS/polyacrylamide gels and its composition shown to resemble closely that of the link component of human intestinal mucin [Mantle, Forstner & Forstner (1984) Biochem. J. 224, 345-354]. Carbohydrates were present, including significant (10 mol/100 mol) amounts of mannose, suggesting the presence of N-linked oligosaccharides. Monospecific antibodies prepared against the rat 118,000-Mr component established its tissue localization in intestinal goblet cells. Mucins subjected to SDS/polyacrylamide-gel electrophoresis and Western blots using the same antibody, established that the link components of rat and human intestinal mucin are similar antigenically. Brief exposure (10 min) of native rat mucin to trypsin or Pronase (enzyme/mucin protein, 1:500, w/w) also released a 118,000-Mr component that reacted with the monospecific antibody. Thus the 118,000-Mr component is an integral part of the mucin and, although linked to large glycopeptides by disulphide bonds, this component also has proteinase-sensitive peptide bonds, presumably at terminal locations such that brief treatment with proteinases releases the molecule in a reasonably intact form. Under physiological conditions, therefore, one might expect that, after mucin is secreted into the intestinal lumen, luminal proteinases would rapidly remove the link component, thereby causing the mucin to depolymerize.
Fahim
R E
RE
Department of Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada.
Specian
R D
RD
Forstner
G G
GG
Forstner
J F
JF
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins; D005455:Fluorescent Antibody Technique; D007124:Immunoenzyme Techniques; D007421:Intestine, Small / Q000032:analysis*; D009077:Mucins; D010446:Peptide Fragments / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D011509:Proteoglycans; D051381:Rats; D000099249:Proteoglycan Link Protein
D000596:Amino Acids; D002241:Carbohydrates; D016326:Extracellular Matrix Proteins; D009077:Mucins; D010446:Peptide Fragments; D011506:Proteins; D011509:Proteoglycans; D000099249:Proteoglycan Link Protein