Phosphomannomutase activity in congenital disorders of glycosylation type Ia determined by direct analysis of the interconversion of mannose-1-phosphate to mannose-6-phosphate by high-pH anion-exchange chromatography with pulsed amperometric detection

• Published in: Analytical biochemistry 2003-06, Vol.317 (1), p.12-18
• Main Authors: Orvisky, E., Stubblefield, B., Long, R.T., Martin, B.M., Sidransky, E., Krasnewich, D.
• Format: Article
• Language: English
• Subjects: Anions; Carbohydrate Metabolism, Inborn Errors - enzymology; Carbohydrate Metabolism, Inborn Errors - genetics; Carbohydrate Metabolism, Inborn Errors - metabolism; Cell Line; Chromatography, Ion Exchange - methods; Congenital disorders of glycosylation; Fibroblasts - cytology; Fibroblasts - enzymology; Glycosylation; Heterozygote; HPAEC-PAD; Humans; Hydrogen-Ion Concentration; Lymphocytes - cytology; Lymphocytes - enzymology; mannose-1-phosphate; Mannosephosphates - analysis; Mannosephosphates - metabolism; Phosphomannomutase activity; Phosphotransferases (Phosphomutases) - deficiency; Phosphotransferases (Phosphomutases) - genetics; Phosphotransferases (Phosphomutases) - metabolism; Sensitivity and Specificity
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/S0003-2697(03)00109-X

Congenital disorders of glycosylation (CDG) are a group of multisystemic disorders resulting from defects in the synthesis and processing of N-linked oligosaccharides. The most common form, CDG type Ia (CDG-Ia), results from a deficiency of the enzyme phosphomannomutase (PMM). PMM converts mannose 6-phosphate (man-6-P) to mannose-1-phosphate (man-1-P), which is required for the synthesis of GDP-mannose, a substrate for dolichol-linked oligosaccharide synthesis. The traditional assay for PMM, a coupled enzyme system based on the reduction of NADP + to NADPH using man-1-P as a substrate, has limitations in accuracy and reproducibility. Therefore, a more sensitive, direct test for PMM activity, based on the detection of the conversion of man-1-P to man-6-P by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), was developed. Using this assay, the activity of PMM was markedly deficient in fibroblasts and lymphoblasts from 23 patients with CDG-Ia (range 0–15.3% of control, average 4.9±4.7%) and also decreased in seven obligate heterozygotes (range 33.0–72.0% of control, average 52.2±14.7%). Unlike the spectrophotometric method, there was no overlap in PMM activity among patients, obligate heterozygotes, or controls. Thus, the PMM assay based on HPAEC-PAD has increased utility in the clinical setting, and can be used, together with transferrin isoelectric focusing, to diagnose patients with CDG-Ia and to identify heterozygotes when clinically indicated.