Cloning and cDNA sequence of a bovine submaxillary gland mucin-like protein containing two distinct domains.
A lambda gt11 cDNA library prepared from bovine submaxillary gland mRNA was screened with polyclonal anti-apo-bovine submaxillary mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. One of the positive clones had a 1.8 kilobase (kb) cDNA insert and coded for an incomplete protein. A 2.0-kb cDNA clone was isolated by rescreening the library with the 1.8-kb cDNA. Nucleotide sequencing of the full-length 2.0-kb cDNA revealed an open reading frame that coded for a 563-amino acid protein. A striking feature of the cloned protein is the skewed distribution of the amino acids, most notably that of the hydroxy amino acids and cysteine. The amino-terminal domain of 339 residues is very rich in threonine, serine, and glycine and poor in cysteine, aspartic acid, tyrosine, phenylalanine, and tryptophan. In contrast, the carboxyl-terminal domain of 224 residues is rich in cysteine, aspartic acid, tyrosine, lysine, and asparagine and relatively poor in threonine, serine, and glycine. A search of the protein data bank for homologies to the deduced amino acid sequence revealed statistically significant matches to several proteins, including the porcine submaxillary apomucin fragment. The cysteine-rich domain by itself was not statistically homologous with any of the registered polypeptide sequences. RNA blot analysis using DNA probes corresponding to the mucin-like and cysteine-rich regions detected a nearly identical pattern of transcripts, demonstrating that the characterized clones are not artifacts of cDNA library construction. The blots also showed the presence of polydisperse transcripts in bovine submaxillary gland but no detectable hybridization signals in liver or brain RNA.
Bhargava
A K
AK
Department of Biological Chemistry, Pennsylvania State University, Hershey, PA 17033.
Woitach
J T
JT
Davidson
E A
EA
Bhavanandan
V P
VP
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000235:genetics; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D003545:Cysteine; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009077:Mucins / Q000235:genetics*; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013363:Submandibular Gland / Q000378:metabolism*
D001059:Apoproteins; D009077:Mucins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D004247:DNA; D003545:Cysteine