Purification, molecular cloning, and sequencing of salivary cystatin SA-1.
A "long form" salivary thiol protease inhibitor, designated cystatin SA-I, was purified to homogeneity from human submandibular-sublingual saliva by sequential gel filtration and ion-exchange chromatography. Automated peptide sequencing data revealed that cystatin SA-I shares sequence homologies with salivary cystatin SN, except that it contains an additional octapeptide at its NH2 terminus. To further characterize the molecular basis of salivary cystatin diversity, a mixed-base oligonucleotide probe corresponding to a region within the NH2-terminal sequence of the salivary cystatins was synthesized. This probe was used to screen a portion of a human submandibular gland cDNA library. The cDNA insert of a clone, designated pBR HSMSF 10G5.1, carried the entire peptide coding sequence of cystatin SA-I. The secretory peptide signal coding sequence was immediately followed by a sequence encoding the eight amino acid residues found at the NH2 terminus of purified cystatin SA-I. To estimate the number of genes encoding cystatins in the human genome, fragments of the pBR HSMSF 10G5.1 insert were used as probes in Southern blot analyses of human genomic DNA. These analyses revealed that the human genome carries 4-7 homologous cystatin genes. Collectively, our data suggest that some of the diversity in salivary cystatins could be generated by expression of different members of a multigene family and by posttranslational proteolytic cleavage of NH2-terminal regions (cystatin SA-I to cystatin SN).
Al-Hashimi
I
I
Department of Oral Biology, School of Dental Medicine, State University of New York, Buffalo 14214.
Dickinson
D P
DP
Levine
M J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D015891:Cystatins; D004247:DNA / Q000235:genetics; D004262:DNA Restriction Enzymes; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping; D011480:Protease Inhibitors; D012463:Saliva / Q000378:metabolism; D055333:Salivary Cystatins; D012471:Salivary Proteins and Peptides / Q000235:genetics* / Q000302:isolation & purification
C525171:CST1 protein, human; C525172:CST2 protein, human; D015891:Cystatins; D011480:Protease Inhibitors; D055333:Salivary Cystatins; D012471:Salivary Proteins and Peptides; D004247:DNA; D004262:DNA Restriction Enzymes