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.
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.
Clinical Neuroscience Branch, NIMH, National Institutes of Health, Bethesda, MD, USA.
E
E
Orvisky
B
B
Stubblefield
R T
RT
Long
B M
BM
Martin
E
E
Sidransky
D
D
Krasnewich
D000838:Anions; D002239:Carbohydrate Metabolism, Inborn Errors / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D006031:Glycosylation; D006579:Heterozygote; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008214:Lymphocytes / Q000166:cytology / Q000201:enzymology; D008360:Mannosephosphates / Q000032:analysis / Q000378:metabolism*; D017875:Phosphotransferases (Phosphomutases) / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity
D000838:Anions; D008360:Mannosephosphates; C047217:mannose 1-phosphate; C027693:mannose-6-phosphate; D017875:Phosphotransferases (Phosphomutases); C052799:phosphomannomutase