Human α-galactosidase gene expression: significance of two peptide regions encoded by exons 1–2 and 6

Abstract

Two proteins with α-galactosidase activity, α-galactosidase A (α-GalA) and α-galactosidase B (α-GalB, or α- N-acetylgalactosaminidase; α-NAGA) have a high homology of amino-acid sequence. Point mutations of the α-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 α-GalA cDNA were partly substituted by the corresponding regions of α-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 α-GalA antigenicity. Their kinetic properties toward 4-methylumbelliferyl α-galactopyranoside were compared with those of α-GalA. The catalytic activity was slightly low in CMB12, decreased to 1 2 in CMB6, and restored to a significant degree in CMB126. K m was more than 4-fold higher for CMB6 and CMB126 than for α-GalA. The pH optimum was 4.0 for both CMB12 and α-GalA, 4.8 for CMB6, and 4.6 for CMB126 and α-GalB. The catalytic activity was inhibited most by galactosamine in CMB6, and less in α-GalB, CMB126, α-GalA and CMB12 in decreasing order. The 50% inhibition concentrations of melibiose (Galα1-6Glc) and methyl α-galactopyranoside were 2.5- to 3-fold higher for CMB126 than for α-GalA. These results indicate that the low affinity of CMB126 to the substrate was caused by a reduced affinity to terminal α-linked galactose. We conclude that (1) the two regions encoded by exons 1–2 and 6 contribute to the α-galactosidic cleavage, and (2) an increase in K m of CMB6 or CMB126, with chimeric substitutions at the exon 6 region, was caused by a loss of affinity toward terminal α-linked galactose.