Genomic Cloning,Physical Mapping,and Expression of Human Type 2 Cystatin Genes

Abstract

Humans carry one gene encoding cystatin C and six to eight genes with homology to an S-like cystatin hybridization probe. However, the precise composition and organization of the cystatin gene family remains to be established. Further, the pattern of tissue-specific expression has not been fully defined. We have previously shown that the type 2 cystatin genes are clustered together in a ca. 270 kb region (the CST locus). To determine the structure of this region, we have sought to clone the entire CST locus. Our approach has been to isolate cosmid and lambda genomic clones carrying cystatin genes and then to use "walk" probes derived from the end regions of these clones to identify other clones, which extend them. To date, we have obtained over 320 kb of distinct sequences. Based on restriction maps, sequencing, and hybridization analyses, we have identified eight apparently nonallelic copies of cystatin genes. These include one gene for cystatin C, four closely related genes encoding S-like cystatins, and three genes encoding relatively divergent sequences. Complete assembly of these clones into an unambiguous contiguous sequence is hampered by the presence of flanking locus-specific repetitive-like sequences. RNase protection assays used to characterize the tissue-specific patterns of expression showed that cystatin C is expressed at modest, comparable levels in all tissues examined, whereas expression of the CST 1 gene, encoding cystatin SA-I, was found to be restricted to a small subset of tissues, with the highest level in the submandibular gland. The cystatin gene family, therefore, appears to have evolved by tandem gene duplication, followed by the acquisition of control elements influencing the location and level of expression. The cystatin gene family is, thus, a potentially powerful system for the future study of mechanisms of gene regulation in human salivary glands.

References

Abrahamson, M., A.J. Barrett, G. Salvesen, and A. Grubb: Isolation of Six Cysteine Proteinase Inhibitors from Human Urine. J. Biol. Chem. 261:11282-11289 (1986).

Al-Hashimi, I., D.P. Dickinson, and M.J. Levine: Purification, Molecular Cloning, and Sequencing of Salivary Cystatin SA-I. J. Biol. Chem. 263:9381-9387 (1988 ).

Barrett, A.J., N.D. Rawlings, M.E. Davies, W. Machleidt, G. Salvesen, and V. Turk: Cysteine Proteinase Inhibitors of the Cystatin Superfamily. In: Proteinase Inhibitors, pp. 515-569. (A. J. Barrett and G. Salvesen, Eds.) Elsevier Science Publishers, Cambridge (1986 ).

Bieth, J.G.: Possible Biological Functions of Protein Proteinase Inhibitors. In: Cysteine Proteinases and their Inhibitors, pp. 694-703. (V. Turk, Ed.) Walter de Gruyter & Co., Berlin (1986).

Bird, A.P.: CpG Islands as Gene Markers in the Vertebrate Nucleus. Trends Genet. 3:342-347 (1987).

Dickinson, D.P., M. Thiesse, L.D. Dempsey, and S.J. Millar: Human S-like Cystatin Genes Are Clustered at Single Locus. J. Dent. Res. (Spec. Iss.) 70: No. 1875 (1991).

Eisenhauer, D.A., R. Hutchinson, T. Javed, and J.K. McDonald: Identification of a Cathepsin B-like Protease in the Crevicular Fluid of Gingivitis Patients. J. Dent. Res. 62:917-921 (1983).

Evans, G.A. and G.M. Wahl: Cosmid Vectors for Genomic Walking and Rapid Restriction Mapping. In: Methods in Enzymology, Vol. 152, pp. 604-610 (S. L. Berger and A. R. Kimmel, Eds.) Academic Press, Inc., San Diego (1987).

Fritz, H., M. Jochum, K.H. Duswald, H. Dittmer, H. Kortmann, S. Neumann, and H. Lang: Granulocyte Proteinases as Mediators of Unspecific Proteolysis in Inflammation: A Review. In: Cysteine Proteinases and Their Inhibitors, pp. 785-807. (V. Turk, Ed.) Walter de Gruyter & Co., Berlin (1986).

10.

Isemura, S., E. Saitoh, K. Sanada, M. Isemura, and S. Ito: Cystatin S and the Related Cysteine Proteinase Inhibitors in Human Saliva. In: Cysteine Proteinases and Their Inhibitors, pp. 497-505. (V. Turk, Ed.) Walter de Gruyter & Co., Berlin (1986).

11.

Lah, T., U. Skaleric, J. Babnik, and V. Turk: Detection of Cathepsin L-like Proteinase and Cathepsin D in Gingival Fluid. J. Periodont. Res. 21:504-509 (1986).

12.

MacDonald, R.J., G.H. Swift, A.E. Przybyla, and J.M. Chirgwin: Isolation of RNA Using Guanidinium Salts. In: Methods in Enzymology, Vol. 152, pp. 604-610 (S. L. Berger and A. R. Kimmel, Eds.) Academic Press, Inc., San Diego ( 1987).

13.

Mort, J.S., A.D. Recklies, and A.R. Poole: Extracellular Presence of the Lysosomal Proteinase Cathepsin B in Rheumatoid Synovium and Its Activity at Neutral pH. Arthritis Rheum. 27:509-515 (1984).

14.

Rawlings, N.D. and A.J. Barrett: Evolution of Proteins of the Cystatin Superfamily. J. Mol. Evol. 30:60-71 (1990).

15.

Sabatini, L.M., T.F. Warner, E. Saitoh, and E.A. Azen: Tissue Distribution of RNAs for Cystatins, Histatins, Statherin, and Proline-Rich Salivary Proteins in Humans and Macaques. J. Dent. Res. 68:1138-1145 (1989).

16.

Saitoh, E., L.M. Sabatini, R.L. Eddy, T.B. Shows, E.A. Azen, S. Isemura, and K. Sanada: The Human Cystatin C Gene (CST 3) Is a Member of the Cystatin Gene Family Which Is Localized on Chromosome 20. In: Biochemical and Biophysical Research Communications, Vol. 162, No. 3, pp. 1324-1331 Academic Press, Inc., San Diego (1989).

17.

Saitoh, E., H.S. Kim, O. Smithies, and N. Maeda: Human Cysteine-Proteinase Inhibitors: Nucleotide Sequence Analysis of Three Members of the Cystatin Gene Family. Gene 61:329-338 (1987).

18.

Sambrook, J., E.F. Fritsch, and T. Maniatis: Molecular Cloning, a Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989).

19.

Thiesse, M., S.J. Millar, and D.P. Dickinson: Physical Mapping by PFGE Identifies a 270 kb Region on Human Chromosome 20 That Contains a Cluster of Type 2 Cystatin Genes. (Submitted).

20.

Tryggvason, K., M. Hoyhtya, and T. Salo: Proteolytic Degradation of Extracellular Matrix in Tumor Invasion. Biochim. Biophys. Acta 907:191-217 (1987).