Three dimensional structural studies of α- N-acetylgalactosaminidase (α-NAGA) in α-NAGA deficiency (Kanzaki disease): different gene mutations cause peculiar structural changes in α-NAGAs resulting in different substrate specificities and clinical phenotypes

Kanzaki disease (OMIM#104170) is attributable to a deficiency in α- N-acetylgalactosaminidase (α-NAGA; E.C.3.2.1.49), which hydrolyzes GalNAcα1- O-Ser/Thr. Missense mutations, R329W or R329Q were identified in two Japanese Kanzaki patients. Although they are on the same codon, the clinical manifestation was more severe in R329W because an amino acid substitution led to protein instability resulting in structural change, which is greater in R329W than in R329Q. To examine whether the different clinical phenotypes are attributable to the two mutations. Plasma α-NAGA activity and urinary excreted glycopeptides were measured and three-dimensional models of human α-NAGA and its complexes with GalNAcα1- O-Ser and GalNAcα1- O-Thr were constructed by homology modeling. Residual enzyme activity was significantly higher in the R329Q- than the R329W mutant (0.022 ± 0.005 versus 0.005 ± 0.001 nmol/h/ml: p < 0.05); the urinary ratios of GalNAcα1- O-Ser:GalNAcα1- O-Thr were 2:10 and 8:10, respectively. GalNAcα1- O-Ser/Thr fit tightly in a narrow space of the active site pocket of α-NAGA. GalNAcα1- O-Thr requires a larger space to associate with α-NAGA because of the side chain (CH 3 ) of the threonine residue. Our findings suggest that the association of α-NAGA with its substrates is strongly affected by the amino acid substitution at R329 and that the association with GalNAcα1- O-Thr is more highly susceptible to structural changes. The residual mutant enzyme in R329W could not associate with GalNAcα1- O-Thr and GalNAcα1- O-Ser. However, the residual mutant enzyme in R329Q catalyzed GalNAcα1- O-Ser to some extent. Therefore, the urinary ratio of GalNAcα1- O-Ser:GalNAcα1- O-Thr was lower and the clinical phenotype was milder in the R329Q mutation. Structural analysis revealed biochemical and phenotypic differences in these Kanzaki patients with the R329Q and R329W mutation.