IDENTIFICATION OF A ZINC-BINDING CYSTIC-FIBROSIS ANTIGEN IN HUMAN SALIVA BY ZN-65 PROBING AND N-TERMINAL SEQUENCING

Human whole saliva was collected at 4 degrees C into a protein inhibit or cocktail to prevent proteolytic degradation. Saliva was clarified b y centrifugation and fractionated by immobilized metal affinity chroma tography (IMAC) after charging the column with zinc ions. Proteins wit h an affinity for zinc were eluted from the IMAC column, characterized by sodium dodecylsulphate-polyacrylamide gel electrophoresis, and pro bed with (ZnCl2)-Zn-65 after electrotransfer on to polyvinylidenefluor ide membranes. A zinc-binding protein of approx. 11 kDa was characteri zed by N-terminal sequencing followed by a FASTA search of Genbank. Th e first 40 residues were sequenced, of which the first 34 residues wer e used to conduct a FASTA search and yielded an homology >97% coding f or sequences of mRNA of two proteins, a cystic fibrosis antigen with a n M-r of 10,938 and a calcium-binding inflammatory protein MRP8 with a n M-r of 10,835 expressed in chronic inflammation. The identity of the last 15 residues of the sequence and the likelihood that the protein is secreted via saliva indicates that the 11-kDa protein is the cystic fibrosis antigen, a protein not previously reported in saliva. (C) 19 97 Published by Elsevier Science Ltd. All rights reserved.