This proposal concerns elucidation of the primary structure of keratinocyte transglutaminase and localization of posttranslational fatty acid and phosphate modifications. First, the complete nucleotide sequence of the cDNA will be obtained. Positive cDNA clones have been selected in an oligo dT-primed lambda gt 11 library with a polyclonal antiserum raised to the purified enzyme. With these probes, the library will be rescreened to identify longer clones covering most of the message sequence. As necessary, new libraries will be prepared with random primers or by primer extension using synthetic oligonucleotides. Genomic clones will then be selected and sequenced, including up to 2 kb of 5'-flanking DNA. To assist in planning the cDNA sequencing, the size of the transglutaminase mRNA will be estimated by northern blot analysis. Cell type specificity of expression of the message will be examined as well as regulation of message levels in keratinocytes by physiological agents known to modulate greatly the expression of the enzyme (retinoids, hydrocortisone, calcium). The transglutaminase amino acid sequence will be deduced from the cDNA nucleotide sequence to assist with determining the orientation of the enzyme in the membrane and sites of posttranslational modification. An anchorage region remaining in the membrane after trypsin release of an active 80 kd fragment will be purified and the N-terminal amino acid sequence obtained. Peptides containing the fatty acid and phosphate modifications will be purified (primarily by HPLC techniques) and sequenced. In addition, the degree of phosphorylation of the transglutaminase will be quantitated. The results of this work will provide a basis for elucidating (i) the role of keratinocyte transglutaminase in cross- linked envelope formation from a molecular perspective (orientation in the membrane, interaction with substrate proteins), (ii) physiological regulation of the enzyme expression by molecular biological techniques, (iii) molecular mechanisms of keratinocyte reprogramming in pathological conditions, and (iv) evolution of transglutaminases.