title: Three dimensional structural studies of alpha-N-acetylgalactosaminidase (alpha-NAGA) in alpha-NAGA deficiency (Kanzaki disease): different gene mutations cause peculiar structural changes in alpha-NAGAs resulting in different substrate specificities and clinical phenotypes.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Dermatology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. takurok@m2.kufm.kagoshima-u.ac.jp
authors.forename: Takuro
authors.initials: T
authors.lastname: Kanekura
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sakuraba
authors.forename: Fumiko
authors.initials: F
authors.lastname: Matsuzawa
authors.forename: Seiichi
authors.initials: S
authors.lastname: Aikawa
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Doi
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Hirabayashi
authors.forename: Noriko
authors.initials: N
authors.lastname: Yoshii
authors.forename: Tomoko
authors.initials: T
authors.lastname: Fukushige
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Kanzaki
mesh_terms: D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D005260:Female; D005838:Genotype; D006027:Glycosides / Q000652:urine; D006801:Humans; D008875:Middle Aged; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D048809:alpha-N-Acetylgalactosaminidase / Q000737:chemistry* / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism
chemical_list: D015295:Antigens, Tumor-Associated, Carbohydrate; D006027:Glycosides; C040286:Tn antigen; C482722:NAGA protein, human; D048809:alpha-N-Acetylgalactosaminidase