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Profilaggrin is a major protein component of the keratohyalin granules of mammalian epidermis. It is initially expressed as a large polyprotein precursor and is subsequently proteolytically processed into individual functional filaggrin molecules. We have isolated genomic DNA and cDNA clones encoding the 5'- and 3'-ends of the human gene and mRNA. The data reveal the presence of likely "CAT" and "TATA" sequences, an intron in the 5'-untranslated region, and several potential regulatory sequences. While all repeats are of the same length (972 bp, 324 amino acids), sequences display considerable variation (10-15%) between repeats on the same clone and between different clones. Most variations are attributable to single-base changes, but many also involve changes in charge. Thus, human filaggrin consists of a heterogeneous population of molecules of different sizes, charges, and sequences. However, amino acid sequences encoding the amino and carboxyl termini are more conserved, as are the 5' and 3' DNA sequences flanking the coding portions of the gene. The presence of unique restriction enzyme sites in these conserved flanking sequences has enabled calculations on the size of the full-length gene and the numbers of repeats in it: depending on the source of genomic DNA, the gene contains 10, 11, or 12 filaggrin repeats that segregate in kindred families by normal Mendelian genetic mechanisms. This means that the human profilaggrin gene system is also polymorphic with respect to size due to simple allelic differences between different individuals. The amino- and carboxyl-terminal sequences of profilaggrin contain partial or truncated repeats with unusual un-filaggrin-like sequences on the termini.(ABSTRACT TRUNCATED AT 250 WORDS)

The transglutaminase 3 enzyme is widely expressed in many tissues including epithelia. We have shown previously that it can bind three Ca 2؉ ions, which in site one is constitutively bound, while those in sites two and three are acquired during activation and are required for activity. In particular, binding at site three opens a channel through the enzyme and exposes two tryptophan residues near the active site that are thought to be important for enzyme reaction. In this study, we have solved the structures of three more forms of this enzyme by x-ray crystallography in the presence of Ca 2؉ and/or Mg 2؉ , which provide new insights on the precise contribution of each Ca 2؉ ion to activation and activity. First, we found that Ca 2؉ ion in site one can be exchanged with difficulty, and it has a binding affinity of K d ‫؍‬ 0.3 M (⌬H ‫؍‬ ؊6.70 ؎ 0.52 kcal/mol), which suggests it is important for the stabilization of the enzyme. Site two can be occupied by some lanthanides but only Ca 2؉ of the Group 2 family of alkali earth metals, and its occupancy are required for activity. Site three can be occupied by some lanthanides, Ca 2؉ , or Mg 2؉ ; however, when Mg 2؉ is present, the enzyme is inactive, and the channel is closed. Thus Ca 2؉ binding in both sites two and three cooperate in opening the channel. We speculate that manipulation of the channel opening could be controlled by intracellular cation levels. Together, these data have important implications for reaction mechanism of the enzyme: the opening of a channel perhaps controls access to and manipulation of substrates at the active site. Transglutaminases (TGases)1 are ubiquitous enzymes that are used widely in biology for many different purposes. There are nine different genes for TGases in the human (1-5). Typically, TGases recognize and activate a protein-bound Gln residue by formation of a thiol-acyl intermediate form. The recognition of a Gln residue may be highly specific, such as is apparently the case of the factor XIIIa, TGase 3, and TGase 4 enzymes, or with rather low specificity as for the TGase 2 enzyme. Next, this acyl intermediate is approached by a nucleophilic second substrate, which transfers onto the Gln residue. Commonly, the nucleophile is water, resulting in the net deamidation of a target Gln residue. If the nucleophile is the ⑀-NH 2 of a protein-bound Lys residue, an isopeptide ⑀-(␥-glutamyl)lysine cross-link is formed. As this cannot be cleaved in animal cells, controlled TGase activity thereby provides an efficient way for the formation of stable, insoluble macromolecular complexes. Other nucleophiles used include polyamines (to form mono-or bi-substituted/cross-linked adducts) or -OH groups to form ester linkages (as in the case of the membranebound TGase 1 enzyme to link epidermis-specific ceramides required for barrier function) (6). The TGase 2 enzyme can bind GTP nucleotides, and there is a reciprocal relationship between this binding and transamidation reactivity (7-9), presumably because the GTP binds in the vicinity of where c...

Intermediate filaments (IF) have been recognized as ubiquitous components of the cytoskeletons of eukaryotic cells for 25 yr. Historically, the first IF proteins to be characterized were those from wool in the 1960s, when they were defined as low sulfur keratins derived from “microfibrils.” These proteins are now known as the type Ia/type IIa trichocyte keratins that constitute keratin IF of several hardened epithelial cell types. However, to date, of the entire class of >40 IF proteins, the trichocyte keratins remain the only ones for which efficient in vitro assembly remains unavailable. In this paper, we describe the assembly of expressed mouse type Ia and type IIa trichocyte keratins into IF in high yield. In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins. However, when oxidized in vitro, several intermolecular disulfide bonds form and the molecular alignments rearrange into the pattern shown earlier by x-ray diffraction analyses of intact wool. We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF. Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.