Excessive Glycine Loop Variations in the Keratin 10 Tail Domain and Implications for Skin Fragility.

Glycine loops in proteins: their occurence in certain intermediate filament chains, loricrins and single-stranded RNA binding proteins

A Large Mutational Study in Pachyonychia Congenita

Monoallelic Large Intragenic KRT5 Deletions Account for Genetically Unsolved Cases of Epidermolysis Bullosa Simplex

5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in animals, fungi, and some bacteria. The enzyme belongs to a large family of enzymes that use pyridoxal 5'-phosphate as an essential cofactor. We previously analyzed the informational content contained in each residue of a conserved glycine loop, which we proposed to form part of the cofactor binding site [Gong, J., & Ferreira, G. C. (1995) Biochemistry 34, 1678-1685]. We found that Gly-142 and -144 contain high informational content, and we identified G144A, G144S, G144T, and G142C as functional mutants. Here, the catalytic parameters, cofactor affinities, and spectral and thermostability properties of these four glycine mutants are determined to examine the function of the glycine loop. In addition, computer models of the glycine loops from the wild-type and mutant enzymes were generated, using glycogen phosphorylase b as the structural template. G144A, G144S, G144T, and G142C displayed lower affinity than the wild-type enzyme for the cofactor, reflected in the 8.5-, 8-, 24.5-, and 15-fold increases, respectively, in the dissociation constant value for binding of the cofactor. While the turnover numbers for G144A, G144S, G144T, and G142C were 43%, 39%, 21%, and 6% of the wild-type value, respectively, the K(m) values for both substrates remained unchanged, with the exception of the G142C K(m)Gly, which showed a 4-fold increase. The UV-visible and CD spectra of Gly-144 mutants were similar to those of the wild type; however, the spectral properties of G142C suggest that this mutant binds the cofactor in a different mode at the active site. G144A, G144S, G144T, and G142C were also found to be less stable than the wild-type enzyme, with the thermotransition temperature, T1/2, determined to be 3.5, 3, 3.5, and 5 degrees C, respectively, lower than that of the wild-type enzyme. Collectively, computer modeling of the wild-type and mutant forms of the ALAS glycine loop and biochemical and spectroscopic characterization of G144A, G144S, G144T, and G142C strongly suggest that the conserved glycine loop in 5-aminolevulinate synthase is a pyridoxal 5'-phosphate cofactor binding motif.

Digenic Inheritance in Epidermolysis Bullosa Simplex

The cornified cell envelope (CE) of terminally differentiated human epidermis is a complex structure consisting of several defined protein constituents. The CE is the most insoluble component of the epidermis due to crosslinking by disulfide bonds as well as isodipeptide bonds that are formed by the action of transglutaminases (TGases). We have recently determined that loricrin is the major component of CE. We now have isolated and characterized its gene and showed that it has a simple structure with a single intron. We also show that the loricrin gene maps to position 1q21, which, coincidentally, is similar to the location of the profilaggrin and involucrin genes. Human loricrin in 26 kDa and consists of three long glycine-serine-cysteine rich sequence domains that contain quasi-repeating peptides and which form the novel glycine loop motif. These are interspersed by lysine+glutamine rich domains involved in isodipeptide crosslinks. The glycine loops are thought to be involved in organization of epidermal proteins and maintenance of the flexibility of the epidermis. By use of PCR analyses, we have found that human loricrin consists of two allelic size variants, due to sequence variations in the second glycine loop domain only, and these variants segregate in the human population by normal Mendelian mechanisms. Furthermore, there are multiple sequence variants within these two size class alleles due to various deletions of 12 bp (4 amino acids) in the major loop of this glycine loop domain. In order to study the expression and role of TGases in the formation of CE, we have isolated and sequenced cDNA and genomic clones encoding the TGase1 enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of eight cDNAs and six genes for intermediate filament (IF) proteins in the cephalochordate Branchiostoma reveals differences in the IF multigene families of lower chordates and the vertebrates.

Although the head and rod domains of intermediate filament (IF) proteins are known to play significant roles in filament assembly, the role of the tail domain in this function is unclear and the available information supports contradictory conclusions. We examined this question by comparing transfection of the same cDNA constructs, encoding vimentins with modified tail domains, into cell lines that do and do not contain endogenous IF proteins. By this approach, we were able to distinguish between the ability of a mutant IF protein to initiate assembly de novo, from that of incorporating into existing filament networks. Vimentins with modifications at or near a highly conserved tripeptide, arg-asp-gly (RDG), of the tail domain incorporated into existing IF networks in vimentin-expressing (vim+) cells, but were assembly-incompetent in cells that did not express IF proteins (vim-). The failure of the RDG mutant vimentins to assemble into filament arrays in vim- cells was reversible by re-introducing a wild-type vimentin cDNA, whereupon both wild-type and mutant vimentins coassembled into one and the same IF network. We conclude that the function of the tail domain of type III IF proteins, and possibly of keratins K8 and K18, in IF assembly is distinct from those of other domains; a region encompassing the RDG tripeptide appears to be important in the assembly process.

We have isolated a cDNA clone from a bovine bladder urothelium library which encodes the smallest intermediate filament (IF) protein known, i.e. the simple epithelial cytokeratin (equivalent to human cytokeratin 19) previously thought to have mol. wt 40,000. This clone was then used to isolate the corresponding gene from which we have determined the complete nucleotide sequence and deduced the amino acid sequence of the encoded protein. This cytokeratin of 399 amino acids (mol. wt 43,893) is identified as a typical acidic (type I) cytokeratin but differs from all other IF proteins in that it does not show the carboxyterminal, non-alpha-helical tail domain. Instead it contains a 13 amino acids extension of the alpha-helical rod. The gene encoding cytokeratin 19 is also exceptional. It contains only five introns which occur in positions corresponding to intron positions in other IF protein genes. However, an intron which in all other IF proteins demarcates the region corresponding to the transition from the alpha-helical rod into the non-alpha-helical tail is missing in the cytokeratin 19 gene. Using in vitro reconstitution of purified cytokeratin 19 we show that it reacts like other type I cytokeratins in that it does not form, in the absence of a type II cytokeratin partner, typical IF. Instead it forms 40-90 nm rods of 10-11 nm diameter which appear to represent lateral associations of a number of cytokeratin molecules. Our results demonstrate that the non-alpha-helical tail domain is not an indispensable feature of IF proteins. The gene structure of this protein provides a remarkable case of a correlation of a change in protein conformation with an exon boundary.

Author response: Intermediate filament network perturbation in the C. elegans intestine causes systemic dysfunctions

BackgroundTanabin, transitin and nestin are type VI intermediate filament (IF) proteins that are developmentally regulated in frogs, birds and mammals, respectively. Tanabin is expressed in the growth cones of embryonic vertebrate neurons, whereas transitin and nestin are found in myogenic and neurogenic cells. Another type VI IF protein, synemin, is expressed in undifferentiated and mature muscle cells of birds and mammals. In addition to an IF-typical α-helical core domain, type VI IF proteins are characterized by a long C-terminal tail often containing distinct repeated motifs. The molecular evolution of type VI IF proteins remains poorly studied.ResultsTo examine the evolutionary history of type VI IF proteins, sequence comparisons, BLAST searches, synteny studies and phylogenic analyses were performed. This study provides new evidence that tanabin, transitin and nestin are indeed orthologous type VI IF proteins. It demonstrates that tanabin, transitin and nestin genes share intron positions and sequence identities, have a similar chromosomal context and display closely related positions in phylogenic analyses. Despite this homology, fast evolution rates of their C-terminal extremity have caused the appearance of repeated motifs with distinct biological activities. In particular, our in silico and in vitro analyses of their tail domain have shown that (avian) transitin, but not (mammalian) nestin, contains a repeat domain displaying nucleotide hydrolysis activity.ConclusionThese analyses of the evolutionary history of the IF proteins fit with a model in which type VI IFs form a branch distinct from NF proteins and are composed of two major proteins: synemin and nestin orthologs. Rapid evolution of the C-terminal extremity of nestin orthologs could be responsible for their divergent functions.

Molecular Heterogeneity of Blistering Disorders: The Paradigm of Epidermolysis Bullosa

PLEC1 Mutations Underlie Adult-Onset Dilated Cardiomyopathy in Epidermolysis Bullosa Simplex with Muscular Dystrophy

The structure of the single gene encoding the cytoplasmic intermediate filament (IF) proteins in non-neuronal cells of the gastropod Helix aspersa is described. Genomic and cDNA sequences show that the gene is composed of 10 introns and 11 exons, spanning greater than 60 kb of DNA. Alternative RNA processing accounts for two mRNA families which encode two IF proteins differing only in their C-terminal sequence. The intron/exon organization of the Helix rod domain is identical to that of the vertebrate type III IF genes in spite of low overall protein sequence homology and the presence of an additional 42 residues in coil 1b of the invertebrate sequence. Intron position homology extends to the entire coding sequence comprising both the rod and tail domains when the invertebrate IF gene is compared with the nuclear lamin LIII gene of Xenopus laevis presented in the accompanying report of Döring and Stick. In contrast the intron patterns of the tail domains of the invertebrate IF and the lamin genes differ from those of the vertebrate type III genes. The combined data are in line with an evolutionary descent of cytoplasmic IF proteins from a nuclear lamin-like progenitor and suggest a mechanism for this derivation. The unique position of intron 7 in the Helix IF gene indicates that the archetype IF gene arose by the elimination of the nuclear localization sequence due to the recruitment of a novel splice site. The presumptive structural organization of the archetype IF gene allows predictions with respect to the later diversification of metazoan IF genes. Whereas models proposing a direct derivation of neurofilament genes seem unlikely, the earlier speculation of an mRNA transposition mechanism is compatible with current results.

Assembly and architecture of invertebrate cytoplasmic intermediate filaments reconcile features of vertebrate cytoplasmic and nuclear lamin-type intermediate filaments