Interaction of heparin with synthetic peptides corresponding to the C-terminal domain of intestinal mucins.
Unlike most other mucins described to date, two intestinal mucins, rat MLP (rat Muc 2) and human MUC2 have a C-terminal tail that is enriched in cationic amino acids. The distribution of charge in each case resembles that of several well known heparin binding proteins. Peptides designated E20-14 and F13-15, corresponding to the C-terminal 14 amino acids of the two mucins, were synthesized and shown to bind 3 H-labelled heparin by a process that was saturable and mediated by strong electrostatic interactions, giving Kd values of 10 (-7) to 10 (-8) M. Using turbidometric analyses and native gel electrophoresis, we observed that peptide-heparin mixtures formed polydisperse aggregates that dissociated with a progressive increase in the concentration of heparin. Under certain conditions heparin protected the peptide from proteolysis by trypsin. Both heparin and dextran sulfate, the latter a highly sulfated synthetic polysaccharide, were potent inhibitors of 3 H-heparin binding to peptide E20-14, while less sulfated glycosaminoglycans were poorly- or non-inhibitory. Mucin in tissue dispersions and homogenates, or purified from rat intestine, did not bind to heparin, and failed to interact with an antibody specific for the peptide E20-14. Both mucin samples however, reacted with antibodies that recognize regions upstream of the C-terminal 14 amino acids. Immunofluorescent localization of E20-14 was confined to the basal perinuclear regions of goblet cells, whereas localization of an antibody to a flanking sequence on the N-terminal side of the C-tail, localized to mature mucin storage granules. These findings suggest that the heparin -binding C-tail of the mucin may be removed at an early stage of biosynthesis. Heparin-mucin complexes, if they form in vivo, are thus likely to be confined to the ER and/or Golgi compartments.
Xu
G
G
Hospital for Sick Children, Toronto, Canada.
Forstner
G G
GG
Forstner
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D005455:Fluorescent Antibody Technique; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D055262:Mucin-2; D009077:Mucins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats
C524625:MUC2 protein, human; C524628:Muc2 protein, rat; D055262:Mucin-2; D009077:Mucins; D010455:Peptides; D011134:Polysaccharides; D006493:Heparin