MOLECULAR-CLONING AND SEQUENCING OF A CANINE TRACHEOBRONCHIAL MUCIN CDNA CONTAINING A CYSTEINE-RICH DOMAIN

To date the complete sequence of only one mammalian mucin cDNA, MUC1, has been reported, although several mucin proteins have been partially characterized. Here we report the nucleotide sequence of a canine tra cheal mucin cDNA containing two potential translation initiation codon s, one translation termination codon and a poly(A) tail. A lambdagt11 cDNA library prepared from canine tracheal epithelial cells was screen ed with polyclonal anti-apo-canine tracheal mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core pro tein. Antibody-positive clones containing overlapping inserts of vario us lengths were purified and used for nucleotide sequencing. Based on the sequencing data, synthetic oligonucleotide primers were constructe d and both ends (5' and 3') of the cDNA were determined. The complete sequence was 3.7 kb and included an open reading frame with coding cap acity for 1118 aa, two translation initiation ATG codons in context wi th Kozak consensus sequences, one polyadenylylation site, and a poly(A ) stretch. The protein was rich in Thr, Pro, Ser, Gly, and Ala and poo r in Tyr, Phe, and Trp. Although tandem repeats of amino acids were ab sent in the deduced canine tracheal mucin sequence, motifs TPTPTP and TTTTPV appeared 13 and 19 times, respectively. The C-terminal region c ontained a Cys-rich domain (although a few Cys residues were also pres ent in the middle of the protein) as has been reported for bovine subm axillary mucin, porcine submaxillary mucin, rat intestinal mucin, huma n intestinal mucin, and frog skin mucin. This suggested that a broad g roup of mucins contain such a Cys-rich domain whose functional signifi cance is yet to be understood. Three potential N-glycosylation sites w ere present in canine tracheal mucin and the amino acid sequence showe d homology with both human tracheal and intestinal mucins. The N-termi nal domain showed more flexibility (probably due to a high number of P ro residues in this region) when analyzed by the University of Wiscons in Genetics Computer Group program package to determine the predicted secondary structure. Evaluation of the transcripts using the canine mu cin cDNA as a probe indicated a polydisperse message with total RNA.