Carbohydrate-deficient glycoprotein syndrome type V: deficiency of dolichyl-P-Glc:Man9GlcNAc2-PP-dolichyl glucosyltransferase

Abstract

Deficiency of dolichyl-P-Glc:Man9GlcNAc2-PP-dolichyl glucosyltransferase is the cause of an additional type of carbohydrate-deficient glycoprotein syndrome (CDGS type V). Clinically this type resembles the classical type Ia of CDGS caused by the deficiency of phosphomannomutase. As a result of the glucosyltransferase deficiency in CDGS type V nonglucosylated lipid-linked oligosaccharides accumulate. The defect is leaky and glucosylated oligosaccharides are found on nascent glycoproteins. The limited availability of glucosylated lipid-linked oligosaccharides explains the incomplete usage of N-glycosylation sites in glycoproteins. This finding is reflected in the presence of transferrin forms in serum that lack one or both of the two N-linked oligosaccharides and the reduction of mannose incorporation to about one-third of control in glycoproteins of fibroblasts.

Figure 1

Pattern of serum transferrin in isoelectric focusing and SDS/PAGE. Samples from a control, a CDGS type Ia reference patient, the study patient, and her parents were subjected to isoelectric focusing (Upper) or SDS/PAGE (Lower), each followed by Western blotting and immunodetection of transferrin. (Upper) 0 to IV indicate transferrin forms with zero or 1–4 neuraminic acids. Slower migrating bands represent higher sialylated forms. (Lower) 0, 1, and 2 indicate tansferrin forms with zero, one, or two oligosaccharide chains.

Figure 2

Glycoprotein-linked oligosaccharides. Control (Left) and patient’s (Right) fibroblasts were metabolically labeled for 30 min with [2-³H]mannose. (Lower) Cultures shown were incubated in the presence of 5 mM N-methyldeoxynojirimycin (MdNM) for 30 min before and during the labeling. [³H]Oligosaccharides were released from glycoproteins with PNGaseF and size-fractionated. (Upper) M₉, G₁, G₂, and G₃ refer to the elution of Man₉GlcNAc₂ and Glc_1–3Man₉GlcNAc₂ standards.

Figure 3

Oligosaccharides in LLO. Fibroblasts from controls (Upper) and the patient (Lower) were metabolically labeled for 30 min with [2-³H]mannose. [³H]Oligosaccharides were released from LLO by mild acid hydrolysis and size-fractionated. M₉, G₁, G₂, and G₃ refer to the elution of Man₉GlcNAc₂ (M₉), G_1–3Man₉GlcNAc₂ (G₁-G₃) standards.

Figure 4

Dol-P-Glc synthase. Microsomal extracts from control (○) and patient’s (▪) fibroblasts were incubated for up to 20 min with UDP-[¹⁴C]Glc in the absence (Upper) or presence of 5 μg of exogenous Dol-P (Lower). Dol-P-Glc was extracted and separated by TLC, and Dol-P-[¹⁴C]Glc was quantified by using a β-scanner.

Figure 5

Dol-P-Glc:Man₉GlcNAc₂-PP-Dol glucosyltransferase. Microsomal extracts from fibroblasts of controls (A-C) and the patient (D) were incubated for 30 min in the presence of [³H]Man₉GlcNAc₂-PP-Dol and Dol-P-[¹⁴C]Glc. After extraction of LLOs, the oligosaccharides were released by mild acid hydrolysis and size-fractionated. (B and C) The column fractions were separately examined for ³H and ¹⁴C-radioactivity. (A) A control extract incubated in the absence of Dol-P-[¹⁴C]Glc. For standards M₉, G₁, G₂, and G₃ see Fig. 3.