Differential expression of three alpha-galactosidase genes and a single beta-galactosidase gene from Aspergillus niger

Abstract

A gene encoding a third alpha-galactosidase (AglB) from Aspergillus niger has been cloned and sequenced. The gene consists of an open reading frame of 1,750 bp containing six introns. The gene encodes a protein of 443 amino acids which contains a eukaryotic signal sequence of 16 amino acids and seven putative N-glycosylation sites. The mature protein has a calculated molecular mass of 48,835 Da and a predicted pI of 4.6. An alignment of the AglB amino acid sequence with those of other alpha-galactosidases revealed that it belongs to a subfamily of alpha-galactosidases that also includes A. niger AglA. A. niger AglC belongs to a different subfamily that consists mainly of prokaryotic alpha-galactosidases. The expression of aglA, aglB, aglC, and lacA, the latter of which encodes an A. niger beta-galactosidase, has been studied by using a number of monomeric, oligomeric, and polymeric compounds as growth substrates. Expression of aglA is only detected on galactose and galactose-containing oligomers and polymers. The aglB gene is expressed on all of the carbon sources tested, including glucose. Elevated expression was observed on xylan, which could be assigned to regulation via XlnR, the xylanolytic transcriptional activator. Expression of aglC was only observed on glucose, fructose, and combinations of glucose with xylose and galactose. High expression of lacA was detected on arabinose, xylose, xylan, and pectin. Similar to aglB, the expression on xylose and xylan can be assigned to regulation via XlnR. All four genes have distinct expression patterns which seem to mirror the natural substrates of the encoded proteins.

FIG. 1

Nucleotide and derived amino acid sequences of aglB. The introns (lowercase letters), putative regulatory sequences (boldface and underlined), signal peptide (lowercase letters), and putative N-glycosylation sites (boldface capital letters) are indicated.

FIG. 2

Alignment of the amino acid sequences of α-galactosidases from A. niger (AglB and AglA; 9), P. purpurogenum (38), T. reesei (AglI; 24), Coffea arabica (51), Cyamopsis tetragonoloba (32), Phaseolus vulgaris (7), Mortierella vinacea (37), Saccharomyces cerevisiae (41), S. paradoxus (29), and Zygosaccharomyces cidri (42). In the consensus sequence, amino acids are depicted which are conserved in at least 7 of the 11 α-galactosidases. Amino acids which are identical in all 11 α-galactosidases are in boldface type.

FIG. 3

Alignment of the amino acid sequences of α-galactosidases from A. niger (AglC; 18), T. reesei (AglII; 24), Thermoanaerobacter ethanolicus (50), Pediococcus pentosaceus (22), Streptococcus mutans (1), and E. coli (4). In the consensus sequence, amino acids are depicted which are conserved in at least four of the six α-galactosidases. Amino acids which are identical in all six α-galactosidases are in boldface type.

FIG. 4

Expression patterns of galactosidase genes from A. niger on different compounds after 4 h of transfer. The 18S rRNA served as an RNA loading control. Percentages are in mass per volume. Abbreviations: glc, glucose; xyl, xylose; gal, galactose.

FIG. 5

Influence of XlnR on the expression of lacA and aglB. Northern blot analysis was performed after 2 h of transfer. The 18S rRNA served as an RNA loading control. Percentages are in mass per volume.