BACKGROUND: Kanzaki disease (OMIM#104170) is attributable to a deficiency in 
alpha-N-acetylgalactosaminidase (alpha-NAGA; E.C.3.2.1.49), which hydrolyzes 
GalNAcalpha1-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.
OBJECTIVE: To examine whether the different clinical phenotypes are attributable 
to the two mutations.
METHODS: Plasma alpha-NAGA activity and urinary excreted glycopeptides were 
measured and three-dimensional models of human alpha-NAGA and its complexes with 
GalNAcalpha1-O-Ser and GalNAcalpha1-O-Thr were constructed by homology modeling.
RESULTS: 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 GalNAcalpha1-O-Ser:GalNAcalpha1-O-Thr were 2:10 and 8:10, 
respectively. GalNAcalpha1-O-Ser/Thr fit tightly in a narrow space of the active 
site pocket of alpha-NAGA. GalNAcalpha1-O-Thr requires a larger space to 
associate with alpha-NAGA because of the side chain (CH3) of the threonine 
residue.
CONCLUSION: Our findings suggest that the association of alpha-NAGA with its 
substrates is strongly affected by the amino acid substitution at R329 and that 
the association with GalNAcalpha1-O-Thr is more highly susceptible to structural 
changes. The residual mutant enzyme in R329W could not associate with 
GalNAcalpha1-O-Thr and GalNAcalpha1-O-Ser. However, the residual mutant enzyme 
in R329Q catalyzed GalNAcalpha1-O-Ser to some extent. Therefore, the urinary 
ratio of GalNAcalpha1-O-Ser:GalNAcalpha1-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.