CHARACTERIZATION AND EXPRESSION OF CDNA CLONES ENCODING HUMAN PROTEASE NEXIN

PROTEASE NEXIN (PN), CDNA CLONES WERE ISOLATED FROM A HUMAN FIBROBLAST LIBRARY USING OLIGOMER PROBES DERIVED FROM THE N-TERMINAL AMINO ACID SEQUENCE OF THE PROTEIN. SEVERAL FULL-LENGTH CLONES WERE CHARACTERIZED AND FOUND TO BE APPROXIMATELY 2,100 BP LONG. THE PN CLONES CONTAINED A 1,260 BP OPEN-READING FRAME THAT DEFINES A 401-AMINO ACID PROTEIN WITH A CHARACTERISTIC LEADER PEPTIDE OF 19 AMINO ACIDS. TWO OF THE FULL-LENGTH CDNA CLONES HAVE BEEN SEQUENCED, AND PNB DIFFER BY TWO AMINO ACIDS, WHICH IS THE RESULT OF INSERTION OF A THREE-BASE CODON, CAG, INTO THE CODING SEQUENCE. THIS THREE-BASE INSERTION PRESERVES THE CODING FRAME, BUT CHANGES THE AMINO ACID SEQUENCE FOR PNA FROM ARG TO THR-GLY IN PNB. THE INSERTION OF THE TRIPLET MAY BE THE RESULT OF AN ALTERNATE SPLICE IN THE 3' REGION OF THE PN GENE. CURRENT STUDIES WILL FOCUS ON DETERMINING THE MECHANISM BY WHICH PNA AND PNB ARISE AND COMPARING THE BIOLOGICAL ACTIVITIES OF THESE PNS. THE STRUCTURE OF THE PN-I GENE WILL BE ANALYZED TO DETERMINE IF THE REGION ENCODING THE INSERTED TRIPLET IS LOCATED AT AN INTRON/EXON JUNCTION AS WOULD BE PREDICTED BY THE MODEL FOR ALTERNATE SPLICING OF THE PRECURSOR MRNA. THE CDNA CODING THE PNA AND PNB WILL BEEXPRESSED IN MAMMALIAN CELLS, AND THE ACTIVITIES OF THESE RECOMBINANT PROTEINS WILL BE STUDIED.