Junctional epidermolysis bullosa (JEB) is an autosomal recessive disorder characterized by blister formation within the dermal-epidermal basement membrane. Genes for the lamina lucida protein, kalinin/laminin 5, have been proposed as candidates for some forms of JEB, based on immunofluorescence analysis recognizing kalinin epitopes. We studied the cDNA of laminin gamma 2 chain for mutations in JEB using heteroduplex analysis. One patient showed a homozygous splice site mutation while another was heterozygous for a deletion-insertion, resulting in a premature termination codon in one allele. Our data implicate mutations in the laminin gamma 2 chain gene (LAMC2) in some forms of JEB.
The expression of human vascular adhesion protein-1 (hVAP-1) is induced at sites of inflammation where extravasation of lymphocytes from blood to the peripheral tissue occurs. We have solved the X-ray structure of hVAP-1, a human copper amine oxidase (CAO), which is distinguished from other CAOs in being membrane-bound. The dimer structure reveals some intriguing features that may have fundamental roles in the adhesive and enzymatic functions of hVAP-1, especially regarding the role of hVAP-1 in inflammation, lymphocyte attachment, and signaling. Firstly, Leu469 at the substrate channel may play a key role in controlling the substrate entry; depending on its conformation, it either blocks or gives access to the active site. Secondly, sugar units are clearly observed at two of the six predicted N-glycosylation sites. Moreover, mutagenesis analysis showed that all of the predicted sites were glycosylated in the protein used for crystallization. Thirdly, the existence of a solvent-exposed RGD motif at the entrance to each active site in hVAP-1 suggests that it may have a functional role.
We report here on the identification and characterization of novel 2-enoyl thioester reductases of fatty acid metabolism, Etr1p from Candida tropicalis and its homolog Ybr026p (Mrf1p) from Saccharomyces cerevisiae. Overexpression of these proteins in S. cerevisiae led to the development of significantly enlarged mitochondria, whereas deletion of the S. cerevisiae YBR026c gene resulted in rudimentary mitochondria with decreased contents of cytochromes and a respiration-deficient phenotype. Immunolocalization and in vivo targeting experiments showed these proteins to be predominantly mitochondrial. Mitochondrial targeting was essential for complementation of the mutant phenotype, since targeting of the reductases to other subcellular locations failed to reestablish respiratory growth. The mutant phenotype was also complemented by a mitochondrially targeted FabI protein from Escherichia coli. FabI represents a nonhomologous 2-enoyl-acyl carrier protein reductase that participates in the last step of the type II fatty acid synthesis. This indicated that 2-enoyl thioester reductase activity was critical for the mitochondrial function. We conclude that Etr1p and Ybr026p are novel 2-enoyl thioester reductases required for respiration and the maintenance of the mitochondrial compartment, putatively acting in mitochondrial synthesis of fatty acids.
Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin-agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380-1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin-biotin technology applications.
The attachment of the marginal gingiva to the tooth surface is mediated by a thin nonkeratinized epithelium termed the junctional epithelium (JE). Ultrastructural studies have revealed that the attachment of the JE to the tooth surface occurs through hemidesmosomes (HD) and a basal lamina-like extracellular matrix termed the internal basal lamina (IBL). We have previously shown that neither type IV collagen nor prototypic laminin, two common components of basement membranes (BM), is present in the IBL between the epithelium and the tooth. In the present study, we show that laminin-5 is a major component of the IBL in both rodent and human tissues. By using in situ hybridization, we also show that the cells of the JE express the LAMC2 gene of laminin-5. In other parts of gingival epithelium, LAMC2 gene expression is less prominent. Our results indicate that the epithelium-tooth interface is a unique structure wherein epithelial cells are induced to secrete a basal lamina containing laminin-5 and no other presently known laminin isoform.
Integrins are a large family of cell surface glycoproteins that mediate cell-cell, cell-extracellular matrix, and matrix-matrix adhesion and transduce bidirectional signals between the cytoplasm and the extracellular matrix or other cells (1). The adhesive function of integrins is important in various physiological processes such as platelet aggregation, inflammation, wound healing, tumor metastasis, cell migration during embryogenesis, viral infections, and other diseases (1, 2). Orthologues of human integrins have been identified in the genomes of birds, amphibians, and bony fish but so far not in the urochordates or in other invertebrates (3).In humans, over 20 different integrin ␣ heterodimers are formed from eight different  subunits (2) and 18 different ␣ subunits (4). The x-ray structure of integrin ␣ V  3 (5) defined much of the heterodimer common to all ␣ integrins, where 12 domains assemble forming the "head" region where ligands bind plus two "tails" leading to the transmembrane helices that anchor integrins to the cell surface. The subunit interface between ␣ V and  3 is mainly formed by a seven-bladed -propeller domain (␣ V ) and an I-like domain ( 3 ). The structure of ␣ V  3 in complex with a cyclic pentapeptide with the arginine-glycineaspartate (RGD) motif showed that the RGD sequence binds into a crevice between the -propeller domain and the I-like domain (6). Upon binding the RGD pentapeptide, the tail regions of ␣ V  3 come closer to each other, and the -propeller domain rotates slightly.Nine integrin ␣ domains have an extra ϳ200-residue inserted domain, the ␣I domain (for a review, see Ref. 7). The ␣I domain is predicted to locate on the side and top of the -propeller domain; currently, the only representative structure of an integrin heterodimer, ␣ V  3 , lacks the ␣I domain (8). In the ␣I domain-containing integrins, the ␣I domain plays a major role in ligand binding, primarily to the metal ion-dependent adhesion site, MIDAS.
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