Human alpha-galactosidase gene expression: significance of two peptide regions encoded by exons 1-2 and 6.
Two proteins with alpha-galactosidase activity, alpha-galactosidase A (alpha-GalA) and alpha-galactosidase B (alpha-GalB, or alpha-N-acetylgalactosaminidase; alpha-NAGA) have a high homology of amino-acid sequence. Point mutations of the alpha-GalA gene have been reported only in the exons 1, 2 or 6. In this study, the exon 1-2 and/or 6 sequences of alpha-GalA cDNA were partly substituted by the corresponding regions of alpha-GalB cDNA, and three chimeric proteins were prepared by the baculovirus expression system: CMB12 with substitution at the exon 1-2 region, CMB6 at the exon 6 region, and CMB126 at both regions. They all preserved alpha-GalA antigenicity. Their kinetic properties toward 4-methylumbelliferyl alpha-galactopyranoside were compared with those of alpha-GalA. The catalytic activity was slightly low in CMB12, decreased to 1/10 in CMB6, and restored to a significant degree in CMB126. Km was more than 4-fold higher for CMB6 and CMB126 than for alpha-GalA. The pH optimum was 4.0 for both CMB12 and alpha-GalA, 4.8 for CMB6, and 4.6 for CMB126 and alpha-GalB. The catalytic activity was inhibited most by galactosamine in CMB6, and less in alpha-GalB, CMB126, alpha-GalA and CMB12 in decreasing order. The 50% inhibition concentrations of melibiose (Gal alpha 1-6Glc) and methyl alpha-galactopyranoside were 2.5- to 3-fold higher for CMB126 than for alpha-GalA. These results indicate that the low affinity of CMB126 to the substrate was caused by a reduced affinity to terminal alpha-linked galactose. We conclude that (1) the two regions encoded by exons 1-2 and 6 contribute to the alpha-galactosidic cleavage, and (2) an increase in Km of CMB6 or CMB126, with chimeric substitutions at the exon 6 region, was caused by a loss of affinity toward terminal alpha-linked galactose.
Ishii
S
S
Department of Clinical Genetics, Tokyo Metropolitan Institute of Medical Science, Japan.
Kase
R
R
Sakuraba
H
H
Fujita
S
S
Sugimoto
M
M
Tomita
K
K
Semba
T
T
Suzuki
Y
Y
D000581:Amidohydrolases; D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000096:biosynthesis; D005091:Exons; D005696:Galactosidases / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010455:Peptides / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; D010455:Peptides; D011993:Recombinant Fusion Proteins; D005696:Galactosidases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase