Abstract
The recent finding that S100A11 is a component of the keratinocyte cornified envelope (CE) (Robinson, N. A., Lapic, S., Welter, J. F., and Eckert, R. L. (1997) J. Biol. Chem. 272, 12035-12046) suggests that S100A11 is a transglutaminase (TG) substrate. In the present study we show that S100A11 forms multimers when cultured keratinocytes are challenged by increased levels of intracellular calcium and that multimer formation is inhibited by the TG inhibitor, cystamine. These S100A11 multimers appear to be incorporated into the CE, as immunoreactive S100A11 is detected in purified envelopes prepared from cultured cells and from foreskin epidermis. To study S100A11 as a transglutaminase substrate, recombinant human S100A11 (rhS100A11) was used in a cell-free cross-linking system. [14C]Putrescine, a primary amine, labels rhS100A11 in a TG-dependent manner. Trypsin digestion of [14C]putrescine-labeled rhS100A11 releases one radiolabeled peptide, Ala98-Lys103. The glutamine residue in this segment, Gln102, is the site of radiolabel incorporation indicating that Gln102 functions as an amine acceptor. The ability of S100A11 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 chromatography profile of trypsin-digested rhS100A11 monomer to that of cross-linked rhS100A11. A unique cross-linked peptide was purified and identified as Met-Ala-Lys3-Ilu-Ser-Ser-Pro-Thr-Glu-Thr-Glu-Arg cross-linked via an Lys3-Gln102 isopeptide bond to Ala-Val-Pro-Ser-Gln102-Lys. These studies show that S100A11 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 epidermis, and that Lys3 and Gln102 are specific sites of cross-link formation.
Highlights
S100A11 Is a Transglutaminase Substrate in Cultured Epidermal Keratinocytes—To study the process of S100A11 crosslinking in keratinocytes, we activated transglutaminase by promoting an increase in the level of intracellular free calcium using high extracellular NaCl [16]
In cells maintained under normal culture conditions, S100A11 monomer is detected in the absence or presence of 20 mM cystamine, a transglutaminase inhibitor
Summary—Our studies show that S100A11, an S100 family protein, is a transglutaminase substrate that is incorporated into the keratinocyte-cornified envelope
Summary
Preparation of Cell Extracts, Conditioned Medium, and Cornified Envelopes from Keratinocytes—Normal human keratinocytes were grown on lethally irradiated 3T3 cells in 50-cm dishes as described previously [15]. The feeder cells were removed and confluent keratinocyte cultures were incubated for 30 min in serum-free media with or without the addition of 20 mM cystamine and cultured for 6 h in the presence or absence of 0.75 M NaCl to induce cornified envelope formation [16]. At the end of the incubation period, the culture medium (6 ml) was collected, transferred into Tris-buffered saline (TBS, 20 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl), and concentrated to 1 ml (per 50-cm culture dish) using an UltraFree-15 centrifugal filter unit (Biomax-5K NMWL, Millipore Corp.). Purified cornified envelope fragments were prepared from cultured keratinocytes or from human foreskin epidermal samples by sonication and extensive washing as described previously [15]
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