Expression pattern of two related cystic fibrosis-associated calcium-binding proteins in normal and abnormal tissues MAUREEN M. WILKINSON 1 , A. BUSUTTIL 2 , CAROLINE HAYWARD 1 , D. J. H. BROCK 1 , JULIA R. DORIN 3 and VERONICA VAN HEYNINGEN 3 * ^ Human Genetics Unit, Department of Medicine, ^Department of Pathology, ^MRC Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK * Author for correspondence Summary This paper reports further study of the identity and function of a protein shown to be elevated in serum from cystic fibrosis (CF) patients and clinically normal heterozygotes. Monoclonal antibodies, specifically recognizing the tentatively named cys- tic fibrosis antigen (CFAg), were produced. Immu- noaffinity purification of CFAg from several sources revealed two components: llxlO 3 and 14xlO 3 M r proteins. cDNA clones corresponding to each protein have been isolated. Data-base com- parisons of the deduced amino acid sequences suggest that both genes encode related but distinct calcium-binding proteins. We propose the name calgranulin A and B, for the l l x l ( r and 14xlO 3 M r components, respectively. It is clear from the as- signment of the calgranulin genes to chromosome 1 that neither is the product of the mutant CF gene, which maps to chromosome 7. We have used the monoclonal antibodies to study the tissue distribution of the two proteins in a wide- ranging immunohistological survey. Where poss- ible the pattern of expression was confirmed by RNA blot analysis. Strong calgranulin expression in granulocytes was confirmed. In addition to myeloid cells, a restricted subset of normal stratified squamous epithelia 'were found to be calgranulin- positive. These included tongue, oesophagus and buccal cells, the last of which has been shown to have altered calmodulin activity in CF patients. Using indirect alkaline phosphatase staining, tissue sections of lung, pancreas and skin (normally con- sidered sites • wher e the CF defect is expressed) were not calgranulin-positive. However, by indirect im- munofluorescence, nasal polyp sections showed weak patchy calgranulin expression in some epi- thelial cells, and stronger, higher frequency ex- pression • whe n such cells • wer e briefly cultured. A number of hyperproliferative, neoplastic or frankly malignant epithelia were found to express the two proteins. Calgranulin expression is a good marker for 'reactive' epithelium in skin and for squamous cell carcinomas of skin, lung and buccal tissues. The calgranulin-positive permanent cell line from a buccal squamous cell carcinoma may prove a suitable tool for unravelling the calgranu- lin-CF relationship. Key words: cystic fibrosis, calcium-binding proteins, immunohistochemistry. Introduction Quantitative measurement of the serum levels of cystic fibrosis antigen (CFAg) was first made possible by the rocket immuno-assay of Manson & Brock (1980), although the presence of an abnormal protein on serum isoelectric focusing was first described by Wilson et al. (1975). CFAg levels were shown to fall into three genotype-dependent classes (Bullock et al. 1982). This pattern, particularly the intermediate levels in clinically unaffected heterozygotes, suggests that the increase of this protein in serum is closely related to the basic defect in this autosomal recessive disease, and that CFAg might Journal of Cell Science 91, 221-230 (1988) Printed in Great Britain © The Company of Biologists Limited 1988 be the product of the CF gene itself. By constructing mouse-human somatic cell hybrids, using parental cells of myeloid origin that express this differentiated cell product, we showed that CFAg is encoded by gene(s) on chromosome 1 (van Heyningen et al. 1985). When the assignment of the disease locus to chromosome 7 was announced (Knowlton et al. 1985; White et al. 1985; Wainwright et al. 1985), it was immediately clear that CFAg is not the product of the CF gene. However, in recessive conditions, knowing the identity and under- standing the function of a secondary abnormality that is expressed in a gene-dose-dependent manner, and there- fore likely to be caused by the underlying disease-causing 221