Primary structure and processing of the Candida tsukubaensisα‐glucosidase

Abstract

The nucleotide sequence of a 4.39‐kb DNA fragment encoding the α‐glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the α‐glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N‐terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the α‐glucosidase precursor is proteolytically processed by removal of an N‐terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63–65 kDa and 50–52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expresion in the heterologus host, the C. tsukubaensisα‐glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensisα‐glucosidase, the rabbit sucrase‐isomaltase complex (proSI) and human lysosomal acid α‐glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.

cDNA cloning and characterisation of an α‐glucosidase gene from potato (Solanum tuberosum L.)

“…The polypeptide encoded by this ORF has an Mr value of 105,400 and an isoelectric point of 5.75. The most significant similarity matches of this amino acid sequence were with members of family 31 of glucosyl transferases (Henrissat and Bairoch, 1993) which includes α-glucosidases from barley (Tibbot and Skadsen, 1996), spinach (Sugimoto et al, 1997), humans (Hoefsloot et al, 1988) and Candida tsukubaenis (Kinsella et al, 1991). The most similar full-length sequences were those of the barley and human α-glucosidases with an overall similarity of approximately 50% and identity of 29%.…”

Identification of the CD45-associated 116-kDa and 80-kDa Proteins as the α- and β-Subunits of α-Glucosidase II

“…Asp-518 has been identified by mutagenesis as the catalytic nucleophile of H-LAG, while Trp-516 has also been shown to be essential for catalytic function (22). These residues, together with flanking sequences, define the catalytic consensus sequence for Family 31 glycosyl hydrolases: (19,(21)(22)(23)(24)(25)(26). As can been seen from the alignments in Fig.…”

Molecular cloning and characterization of a gibberellin-inducible, putative ?-glucosidase gene from barley

“…These studies identify a putative barley a-glucosidase cDNA clone, pAGL.2752, that potentially encodes a polypeptide homologous to human lysosomal a-glucosidase and other members of glycosyl hydrolase family 31 [4,8,28]. The barley enzyme is more homologous to family 31 than to family 13 (maltase and a-amylase) or 15 (glucoamylase) (Fig.…”

“…An alignment between barley ~-glucosidase and ~-glucosidases belonging to glycosyl hydrolase family 31 was performed. The names, polypeptide lengths, regions analyzed and SwissProt accession numbers were as follows: human lysosomal ~-glucosidase (952 amino acids (aa), 189-862, P10253 [18]); rabbit intestinal pro-sucrase-isomaltase complex (1827 aa, 168-835 isomaltase, 1040-1729 sucrase, P07768 [22]); Schwanniomyces occidentalis glucoamylase (958 aa, 130-871, P22861 [8 ]); Candida tsukubaensis ~-glucosidase (1070 aa, 122-975, P29064 [28]) and barley 2-glucosidase (877 aa, 85-764, U22450 submitted).…”

“…Several e-glucosidase genes, and genes for other enzymes with e-l,4-and/or e-l,6-exoglucolytic activity, have been cloned in bacteria [38,54], fungi [8,12,22,28,32], insects [23,48], and mammals [19,22]. These enzymes have a (/~/e)8 structure and fall into three glycosyl hydrolase families, based on amino acid sequence similarities [17,52].…”