IDENTIFICATION OF TRANSGLUTAMINASE-REACTIVE RESIDUES IN S100A11

The recent finding that S100A11 is a component of the keratinocyte cor nified envelope (CE) (Robinson, N. A., Lapic, S., Welter, J. F., and E ckert, R. L. (1997) J. Biol. Chern, 272, 12035-12046) suggests that S1 00A11 is a transglutaminase (TG) substrate, In the present study we sh ow that S100All forms multimers when cultured keratinocytes are challe nged by increased levels of intracellular calcium and that multimer fo rmation is inhibited by the TG inhibitor, cystamine, These S100All mul timers appear to be incorporated into the CE, as immunoreactive S100A1 1 is detected in purified envelopes prepared from cultured cells and f rom foreskin epidermis, To study S100A11 as a transglutaminase substra te, recombinant human S100All (rhS100A11) was used in a cell-free cros s linking system, [C-14]Putrescine, a primary amine, labels rhS100A11 in a TG-dependent manner. Trypsin digestion of [C-14]putrescine-labele d rhS100A11 releases one radiolabeled peptide, Ala(98)-Lys(103). The g lutamine residue in this segment, Gln(102), is the site of radiolabel incorporation indicating that Gln(102) functions as an amine acceptor, The ability of S100All to form multimers indicates that it also has a reactive lysine residue that functions as an amine donor, To identify the reactive residue, we compared the high pressure liquid chromatogr aphy profile of trypsin-digested rhS100A11 monomer to that of cross-li nked rhS100A11, A unique cross linked peptide was purified and identif ied as Met-Ala-Lys(3)-Ilu-Ser-Ser-Pro-Thr-Glu-Thr-Glu-Arg cross-linked via an Lys(3)-Gln(102) isopeptide bond to Ala-Val-Pro-Ser-Gln(102)-Ly s. These studies show that S100All is post-translationally modified by transglutaminase, that it can be cross linked to form multimers, that it is present in CEs from cultured keratinocytes and in vivo epidermi s, and that Lys(3) and Gln(102) are specific sites of cross link forma tion.