Anosmin-1 is the glycoprotein encoded by the KAL1 gene and part of the extracellular matrix, which was fi rst identifi ed as defective in human Kallmann syndrome (KS, characterised by hypogonadotropic hypogonadism and anosmia); biochemically it is a cell adhesion protein. The meticulous biochemical dissection of the anosmin-1 domains has identifi ed which domains are necessary for the protein to bind its different partners to display its biological effects. Research in the last decade has unravelled different roles of anosmin-1 during CNS development (axon pathfi nding, axonal collateralisation, cell motility and migration), some of them intimately related with the cited KS but not only with this. More recently, anosmin-1 has been identifi ed in other pathological scenarios both within (multiple sclerosis) and outside (cancer, atopic dermatitis) the CNS.
The molecular cloning of 1,3-beta-glucanase-encoding genes from different yeast species was achieved by screening genomic libraries with DNA probes obtained by PCR-amplification using oligonucleotides designed according to conserved regions in the EXG1, EXG2 and SSG1 genes from Saccharomyces cerevisiae. The nucleotide sequence of the KlEXG1 (Kluyveromyces lactis), HpEXG1 (Hansenula polymorpha) and SoEXG1 (Schwanniomyces occidentalis) genes was determined. K1EXG1 consists of a 1287 bp open reading frame encoding a protein of 429 amino acids (49,815 Da). HpEXG1 specifies a 435-amino acid polypeptide (49,268 Da) which contains two potential N-glycosylation sites. SoEXG1 encodes a protein of 425 residues (49,132 Da) which contains one potential site for N-linked glycosylation. Expression in S. cerevisiae of KlEXG1, SoEXG1 or HpEXG1 under control of their native promoters resulted in the secretion of active 1,3-beta-glucanases. Disruption of KlEXG1 did not result in a phenotype under laboratory conditions. Comparison of the primary translation products encoded by KlEXG1, HpEXG1 and SoEXG1 with the previously characterized exo-1,3-beta-glucanases from S. cerevisiae and C. albicans reveals that enzymes with this type of specificity constitute a family of highly conserved proteins in yeasts. KlExg1p, HpExg1p and SoExg1p contain the invariant amino acid positions which have been shown to be important in the catalytic function of family 5 glycosyl hydrolases.
The YlEXG1 gene of Yarrowia lipolytica, encoding an exo-1, 3-beta-glucanase, was isolated by screening a genomic library with a DNA probe obtained by PCR amplification, using oligonucleotides designed according to conserved regions in the EXG1, EXG2 and SSG1 genes from Saccharomyces cerevisiae. YlEXG1 consists of a 1263 bp open reading frame encoding a protein of 421 amino acids with a calculated molecular weight of 48 209 Da. Northern blot analysis revealed a unique YlEXG1-specific transcript, 1.4 kb long. A putative pre(signal)-peptide of 15 amino acids is proposed at the N-terminal domain of the primary translation product. The deduced amino acid sequence shares a high degree of homology with exo-1, 3-beta-glucanases from other yeast species, including S. cerevisiae, Kluyveromyces lactis, Pichia angusta and Debaryomyces occidentalis. YlExg1p contains the invariant amino acid positions which have been shown to be important in the catalytic function of family 5 glycosyl hydrolases. Chromoblot analysis indicated that YlEXG1 is located on chromosome VI. Disruption of YlEXG1 did not result in a phenotype under laboratory conditions and did not prevent the yeast-hypha transition. The sequence data reported in this paper have been assigned EMBL Accession No. Z46872.
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