Biochemical and structural analysis of missense mutations in N-acetylgalactosamine-6-sulfate sulfatase causing mucopolysaccharidosis IVA phenotypes

Abstract

Mucopolysaccharidosis IVA (MPS IVA; OMIM#253000), a lysosomal storage disorder caused by a deficiency of N -acetylgalactosamine-6-sulfate sulfatase (GALNS), has variable clinical phenotypes. To date we have identified 65 missense mutations in the GALNS gene from MPS IVA patients, but the correlation between genotype and phenotype has remained unclear. We studied 17 missense mutations using biochemical approaches and 32 missense mutations, using structural analyses. Fifteen missense mutations and two newly engineered active site mutations (C79S, C79T) were characterized by transient expression analysis. Mutant proteins, except for C79S and C79T, were destabilized and detected as insoluble precursor forms while the C79S and C79T mutants were of a soluble mature size. Mutants found in the severe phenotype had no activity. Mutants found in the mild phenotype had a considerable residual activity (1.3-13.3% of wild-type GALNS activity). Sulfatases, including GALNS, are members of a highly conserved gene family sharing an extensive sequence homology. Thus, a tertiary structural model of human GALNS was constructed from the X-ray crystal structure of N -acetylgalacto-samine-4-sulfatase and arylsulfatase A, using homology modeling, and 32 missense mutations were investigated. Consequently, we propose that there are at least three different reasons for the severe phenotype: (i) destruction of the hydrophobic core or modification of the packing; (ii) removal of a salt bridge to destabilize the entire conformation; (iii) modification of the active site. In contrast, mild mutations were mostly located on the surface of the GALNS protein. These studies shed further light on the genotype-phenotype correlation of MPS IVA and structure-function relationship in the sulfatase family.

Identification of a common mutation in mucopolysaccharidosis IVA: correlation among genotype, phenotype, and keratan sulfate

“…One mutation (p.R380S) was associated with a milder phenotype, while 11 other mutations were associated with a severe clinical phenotype of each patient, homozygosity of the mutation, and the expression studies for p.G139S, p.R380S, and p.R386C Sukegawa et al 2000; the current study).…”

“…The p.R386C mutation was found as a homozygous state in four patients from Argentina, Colombia, and Chile, and a heterozygous state in four patients from all four countries. The p.R386C (CGT to TGT) mutation at the CpG dinucleotide, nonconservative, amino acid change generates a large structural alteration of GALNS protein (Sukegawa et al 2000) leading to a severe form of MPS IVA. This allele frequency was found to be highest among unrelated mutant alleles in this study.…”

Mucopolysaccharidosis IVA mutations in Chinese patients: 16 novel mutations

“…The 3D structural model of human GALNS has been constructed by homology modeling of the X-ray crystal structure of human 4S and ASA, 27 although the X-ray crystal structure of GALNS was not available.…”

“…p.W325X could cause the loss of some N-terminal components (the last b-sheet and last two a-helixes) and all C-terminal components (four b-sheets and one a-helix); p.Q422X would lead to the loss of the last C-terminal a-helix. 24,27 Two novel intronic splicing mutations, c.567-1G4T and c.634-1G4A, were the first splicing defects identified in introns 5 and 6. Both mutations affected the 3¢-acceptor site, which is believed to be the most conserved intronic splicing elements.…”

Análise Estrutural de Mutações na Enzima GALNS associadas à Mucopolissacaridose IVA utilizando a Técnica de Modelagem Comparativa

“…54. Sukegawa, K., Nakamura, H., Kato, Z., Tomatsu, S., Montaño, A. M., Fukao, T., & Kondo, N. (2000). Biochemical and structural analysis of missense mutations in Nacetylgalactosamine-6-sulfate sulfatase causing mucopolysaccharidosis IVA phenotypes.…”