Biochemical and Structural Analysis of Keratan Sulphate and Chondroitin Sulphate from Urine of Patients with Mucopolysaccharidosis IVA
Vicente, C. M.1, Coulson-Thomas, V.J.1, Ricci, R.1, Albano, L.M.2, Kim, C.A.2 and Toma, L.1
1Disciplina de Biologia Molecular, Departamento de Bioquímica, UNIFESP.
2Departamento de Pediatria, Instituto da Criança, USP.
Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive lysosomal storage disorder caused by a genetic defect in N-acetylgalactosamine-6-sulfate sulfatase (GALNS, E.C.3.1.6.4, OMIM#253000). This enzyme is one of the sulfatases required to degrade keratan sulphate (KS) and chondroitin sulphate (CS). MPS IVA patients have a broad spectrum of clinical severity, which includes severe bone dysplasia, short trunk dwarfism, coxa valga, odontoid hypoplasia, corneal opacity and preserved inteligence.
This study involves the structural analysis of the KS and CS purified from the urine of MPS IVA patients, comparing with normal individuals.
GAGs were extracted from urine by ion exchange resin Q-Sepharose; fractions containing GAGs were desalted in Sephadex G-25/PD10 column.
KS was isolated after incubation with crude extract of F. hepatinum followed by two gel filtration chromatographies Sephadex G-25 and Sephadex G-10. Through enzymatic incubations with keratanases from Pseudomons sp and Bacillus sp we have demonstrated the presence of the KS otherwise not detected. The main product of this KS is a disaccharide-6- sulfated.
Analysis of CS was done with chondroitinases AC and ABC, two bacterial lyases. The digestion showed a distinct pattern of CS sulfation in MPS IVA patients compared to normal individuals. 52% of patients CS is 6-sulfated (52% DU-GalNAc6S, 30% DU-GalNAc4S, 18% DU-GalNAc or GalNAc6S). This profile contrasts with the normal ones, which presented only 35% of disaccharide-6-sulfated (35%, 44% and 21%, respectivelly).
The accumulation of KS and CS in the lysosomes of MPS IVA patients can be well correlated with the growth delay presented by these patients. Chondroitin-4- sulphate is involved in the process of growth and ossification, whereas chondroitin-6-sulfate establishes along the process and predominates in adult bone. Severe bone deformation as well as growth impairment in MPS IVA patients corroborates with the functional role assigned for both chondroitin sulfates isomers, and show their importance to human growth and bone development.
(Supported by CAPES, CNPq)
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