Identification of two glycoforms of the MUC5B mucin in human respiratory mucus. Evidence for a cysteine-rich sequence repeated within the molecule.
It has been demonstrated previously that respiratory secretions contain three oligomeric, gel-forming mucins; one of these was identified as the product of the MUC5AC gene (1). Here we demonstrate that the other two mucins are glycoforms of the MUC5B gene product. This was accomplished by trypsin treatment of the purified reduced mucin subunit populations and N-terminal sequencing of the liberated peptides. The products of trypsin digestion were separated by gel filtration into high molecular weight mucin glycopeptides and low molecular weight tryptic peptides. The latter were fractionated by reverse phase chromatography, and four of the major peptides were sequenced. Three of these peptides were identical to and contiguous within a 51-amino acid sequence deduced from a cDNA clone (JER57) encoding a portion of the MUC5B mucin. The other peptide is also present within this sequence but showed identity in only 9 of its 10 residues. A polyclonal antiserum raised against one of these peptides was reactive with the two putative MUC5B glycoforms. Analysis of the high molecular weight glycopeptides indicated that the MUC5B subunit contained different types and lengths of glycosylated domains; one domain of Mr 7.3 x 10(5), two domains of Mr 5.2 x 10(5), and a third domain of Mr 2.0 x 10(5). The amino acid composition of the larger two glycopeptides was similar in serine, threonine, and proline content but distinct from that of the smallest glycopeptide. Each of these domains in the mucin subunit is separated by a trypsin-sensitive region, and the relative abundance of the major peptides derived by proteolysis of these regions and their occurrence in a contiguous sequence suggest that they contain a common cysteine-rich motif.
Thornton
D J
DJ
The Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, 2.205, Stopford Building, Manchester M139PT, United Kingdom. Dave.Thornton@man.ac.uk
Howard
M
M
Khan
N
N
Sheehan
J K
JK
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055257:Mucin-5B; D009077:Mucins / Q000737:chemistry*; D009093:Mucus / Q000737:chemistry*; D010449:Peptide Mapping; D011487:Protein Conformation; D012137:Respiratory System / Q000378:metabolism*; D016415:Sequence Alignment; D014357:Trypsin / Q000378:metabolism
C524578:MUC5B protein, human; D055257:Mucin-5B; D009077:Mucins; D014357:Trypsin; D003545:Cysteine