Effects of glycosylation and pH conditions in the dynamics of human arylsulfatase A

Abstract

Arylsulfatase A (ARSA) is a lysosomal sulfatase that catalyzes the hydrolysis of cerebroside sulfate. Its deficiency results in Metachromatic Leukodystrophy, whereas a minor condition called ARSA pseudodeficiency occurs in healthy individuals, which has been associated with the substitution of the glycosylated Asn350 by a Ser and with the loss of the polyadenylation signal. In this work, we have investigated ARSA dynamics employing molecular dynamics simulations in response to (1) different pH’s, as, beyond its natural lysossomal environment, it has been recently identified in cytoplasmatic medium and (2) glycan occupancies, including its normal glycosylation state, presenting three high mannose-type oligosaccharides. Accordingly, four systems were studied considering ARSA under different conditions: (1) nonglycosylated at pH ∼ 7 (ARSA_pH7); (2) non-glycosylated at pH ∼ 5 (ARSA_pH5); (3) triple glycosylated at pH ∼ 5 (ARSA_glyc,pH5); and (4) ARSA-N350S mutant at pH ∼ 5 (ARSA_N350S,pH5). Lowering pH and increasing glycosylation was found to reduce the flexibility of the enzyme. In addition, at acidic pH, the glycosylated enzyme presented a higher secondary conformational stability when compared to its nonglycosylated counterpart, supporting experimental findings on triple glycosylation as the essential state of ARSA. The N350S mutant exhibited a consistent degree of unfolding, which may be related to its in vitro reduced stability. Finally, the obtained data are discussed in the search for structural evidences able to contribute to the understanding of biological activity of ARSA and molecular etiology of ARSA pseudodeficiency, as determined by ARSA-N350S in the absence of polyadenylation defect.

Keywords:

• ARSA
• glycan
• magnesium
• mutation
• sulfatase

Acknowledgments

This study was supported by the Brazilian Funding Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), MCT; by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), MEC, Brasília, DF, Brazil; by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS); and by the Fundo de Incentivo à Pesquisa e Eventos do Hospital de Clínicas de Porto Alegre (FIPE-HCPA).