Role of the cystine-knot motif at the C-terminus of rat mucin protein Muc2 in dimer formation and secretion

Biochem J. 2001 Jul 1;357(Pt 1):203-9. doi: 10.1042/0264-6021:3570203.

Abstract

DNA constructs based on the 534-amino-acid C-terminus of rat mucin protein Muc2 (RMC), were transfected into COS cells and the resultant (35)S-labelled dimers and monomers were detected by SDS/PAGE of immunoprecipitates. The cystine-knot construct, encoding the C-terminal 115 amino acids, appeared in cell lysates as a 45 kDa dimer, but was not secreted. A construct, devoid of the cystine knot, failed to form dimers. Site-specific mutagenesis within the cystine knot was performed on a conserved unpaired cysteine (designated Cys-X), which has been implicated in some cystine-knot-containing growth factors as being important for intermolecular disulphide-bond formation. Dimerization of RMC was effectively abolished. Each cysteine (Cys-1-Cys-6) comprising the three intramolecular disulphide bonds of the cystine knot was then mutated. Dimer formation was impaired in each case, although much less so for the Cys-3 mutant than the others. Abnormal high-molecular-mass, disulphide-dependent aggregates formed with mutations Cys-1, Cys-2, Cys-4 and Cys-5(,) and were poorly secreted. It is concluded that the intact cystine-knot domain is essential for dimerization of the C-terminal domain of rat Muc2, and that residue Cys-X in the knot plays a key role. The structural integrity of the cystine knot, maintained by intramolecular bonds Cys-1-Cys-4, Cys-2-Cys-5 and Cys-3-Cys-6, also appears to be important for dimerization, probably by allowing correct positioning of the unpaired Cys-X residue for stable intermolecular cystine-bond formation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cysteine
  • Cystine*
  • DNA Primers
  • Dimerization
  • Disulfides / analysis
  • Mucin-2
  • Mucins / chemistry*
  • Mucins / genetics
  • Mucins / metabolism*
  • Mutagenesis, Site-Directed
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Serine

Substances

  • DNA Primers
  • Disulfides
  • Muc2 protein, rat
  • Mucin-2
  • Mucins
  • Recombinant Proteins
  • Serine
  • Cystine
  • Cysteine