Dolichol phosphate mannose synthase (DPM1) mutations define congenital disorder of glycosylation Ie (CDG-Ie)

Abstract

Congenital disorders of glycosylation (CDGs) are metabolic deficiencies in glycoprotein biosynthesis that usually cause severe mental and psychomotor retardation. Different forms of CDGs can be recognized by altered isoelectric focusing (IEF) patterns of serum transferrin (Tf). Two patients with these symptoms and similar abnormal Tf IEF patterns were analyzed by metabolic labeling of fibroblasts with ¿2-(3)Hmannose. The patients produced a truncated dolichol-linked precursor oligosaccharide with 5 mannose residues, instead of the normal precursor with 9 mannose residues. Addition of 250 microM mannose to the culture medium corrected the size of the truncated oligosaccharide. Microsomes from fibroblasts of these patients were approximately 95% deficient in dolichol-phosphate-mannose (Dol-P-Man) synthase activity, with an apparent K(m) for GDP-Man approximately 6-fold higher than normal. DPM1, the gene coding for the catalytic subunit of Dol-P-Man synthase, was altered in both patients. One patient had a point mutation, C(274)G, causing an R(92)G change in the coding sequence. The other patient also had the C(274)G mutation and a 13-bp deletion that presumably resulted in an unstable transcript. Defects in DPM1 define a new glycosylation disorder, CDG-Ie.

Figure 1

Tf IEF patterns of control and patients with CDG. Patients deficient in phosphomannomutase (PMM, CDG-Ia) and phosphomannose isomerase (PMI, CDG-Ib) have similar patterns. Patients PY and CH have a prominent increase in disialo transferrin compared with controls. Numbers at right indicate numbers of sialic acid residue per transferrin molecule.

Figure 2

Analysis of [2-³H]mannose-labeled oligosaccharides. (a) Con A-Sepharose analysis of PY oligosaccharides labeled with [2-³H]mannose with (filled squares) or without (open squares) a 250 mM mannose supplement as described in Methods. The control profiles with and without mannose were superimposable, and only 1 (filled triangles) is shown. Results are percentage of the total counts recovered. Oligosaccharides from fraction III of control (b) and patient (c) labeled without mannose supplement were mixed with fluorescent 2-AB–labeled internal standards and fractionated by HPLC. The elution positions of standards Man₅GlcNAc₂-2-AB and Man₉GlcNAc₂-2-AB (1 and 2, respectively) are indicated by the arrows. 2-AB–labeled standards elute earlier than the corresponding free oligosaccharides. Arrowheads indicate position of [2-³H]Man₅GlcNAc₂.

Figure 3

Exogenous mannose corrects the size of LLO from patient PY. Cells from control or patient PY were labeled for 1 hour with [2-³H]mannose in medium containing 0.5 mM glucose or 0.5 mM glucose supplemented with 250 mM mannose. The LLO was isolated, hydrolyzed from the lipid, and analyzed by HPLC to separate the oligosaccharides based on their size. Arrows indicate the elution positions of [2-³H]Man₅GlcNA₂ (M5N2) and Glc₃[2-³H]Man₅GlcNAc₂ (G3M9N2).

Figure 4

Dol-P-Man synthase activities. (a) Activity of control, CDG-Ic patient, PY, and CH. (b) Dol-P-Man synthesized from control (x), CH (open triangles), and PY (filled diamonds) as a function of GDP-Man concentration. Insert shows the calculated apparent K_m and V_max.

Figure 5

Semiquantitative RT-PCR from total fibroblast RNA. Five microliters of the products from each RNA concentration were loaded on a 1.5% agarose gel and viewed with ethidium bromide. Reaction conditions are described in Methods.