Sequence variations of the CST2 gene related to the polymorphism of salivary cystatin SA

The CST2 locus has two polymorphic alleles, CST2*1 and CST2*2, which produc e cystatin proteins SAI and SA2, respectively (Shintani el al., 1994). The purpose of this study was to define nucleotide sequence variations of the p rotein-coding region of the two alleles. The variations were investigated b y direct sequencing of amplified DNA from individuals with different CST2 p henotypes. The sequence of three exons obtained from DNA of the CST2 1 phen otype was found to be identical to the published sequence of the CST2 gene (Saitoh et al., 1987), whereas two-point mutations were found in the sequen ce obtained from DNA of the CST2 2 phenotype. One of the mutations was a G --> A transition in exon 2, resulting in loss of a commonly occurring AciI restriction site. This mutation resulted in a Gly(59) --> Asp(59) substitut ion in the protein. The other mutation was an A --> T transversion in exon 3, resulting in the generation of a SfaNI restriction site. This mutation a lso produced a Glu(120) --> Asp(120) substitution in the protein. PCR-RFLP assay with AciI and SfaNI restriction enzymes revealed that the two-point m utations were always correlated with cystatin SA polymorphism. The differen ce in the electrophoretic positions of the two proteins, SA1 and SA2, in a basic gel and in an isoelectric focusing gel agreed with the expected mobil ities of the proteins with the SA2 variant at a more anodal position. The C ST2*2 allele is a unique allele, which shows amino acid substitution in one of the most conserved regions responsible for cysteine proteinase inhibito ry activity.