Conserved oligomeric Golgi complex subunit 1 deficiency reveals a previously uncharacterized congenital disorder of glycosylation type II

Abstract

The conserved oligomeric Golgi (COG) complex is a heterooctameric complex that regulates intraGolgi trafficking and the integrity of the Golgi compartment in eukaryotic cells. Here, we describe a patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex. This patient has a defect in both N- and O-glycosylation. Mass spectrometric analysis of the structures of the N-linked glycans released from glycoproteins from the patient's serum revealed a reduction in sialic acid and galactose residues. Peanut agglutinin (PNA) lectin staining revealed a decrease in sialic acids on core 1 mucin type O-glycans, indicating a combined defect in N- and O-glycosylation. Sequence analysis of the COG1 cDNA and gene identified a homozygous insertion of a single nucleotide (2659-2660insC), which is predicted to lead to a premature translation stop and truncation of the C terminus of the Cog1 protein by 80 amino acids. This mutation destabilizes several other COG subunits and alters their subcellular localization and hence the overall integrity of the COG complex. This results in reduced levels and/or altered Golgi localization of alpha-mannosidase II and beta-1,4 galactosyltransferase I, which links it to the glycosylation deficiency. Transfection of primary fibroblasts of this patient with the full length hemagglutinin-tagged Cog1 indeed restored beta-1,4 galactosyltransferase Golgi localization. We propose naming this disorder CDG-II/Cog1, or CDG-II caused by Cog1 deficiency.

Fig. 1.

IEF of serum transferrin and plasma ApoC-III, and PNA lectin staining of fibroblasts. (A) IEF of serum transferrin from a control, the patient under investigation, and a CDG-I patient are shown. The number of negative charges is indicated on the right. (B) IEF of plasma ApoC-III. Serum samples from a healthy control and the patient are shown. The ApoC-III forms with two, one, or zero neuraminic acids are indicated as ApoC-III₂, -III₁, and -III₀, respectively. (C–F) PNA–Alexa Fluor 588 staining of control fibroblasts (C and E) and patient fibroblasts (D and F) before (C and D) and after (E and F) treatment with neuraminidase. All images were obtained at ×20.

Fig. 2.

Identification of a 2559–2660insC mutation in the genomic DNA of the patient. (A) The Western blots of whole-cell lysates from control and patient fibroblasts were quantified by using an Odyssey integrator, and the relative intensities of the bands in the patient's sample are represented as mean ± SD (n = 3). Actin levels were used as a loading control. (B) Sequence alignment of the cDNA fragment from a control, a patient homozygous for the 2559–2660insC mutation, and the heterozygous parents. The arrow indicates the location of the mutation. (C) Indirect immunofluorescence staining of control (Upper) and patient (Lower) fibroblasts using antibodies for Cog1 (Left) and GM130 (Center), and the merged images (Right). The images of control and patient fibroblast were collected with the same laser power and settings. (Scale bar, 30 μm.) (D) Western blot of membrane and cytosol fractions from control (C) and patient (P) fibroblasts.

Fig. 3.

Expression of Golgi α-mannosidase II (ManII), β-1,4 galactosyltransferase I (B1,4 GalT1), GPP130, and GM130 in control and patient fibroblast: intracellular distribution (A) and steady-state levels of expression (B). (A Upper) Control and patient fibroblasts were processed for double indirect immunofluorescence microscopy by using antibodies against the indicated proteins. Images were collected under identical confocal settings. (Lower) Quantification of immunofluorescence intensities in the patient fibroblasts as a percentage of control. Results are mean ± SD, n = 10 cells. (Scale bar, 30 μm.) (B) Western blots of whole-cell lysates from control (C) and patient (P) fibroblasts. Actin levels were used as a loading control.

Fig. 4.

Rescue of β-1,4 galactosyltransferase I levels in patient fibroblasts by transient transfection with HA-COG1 cDNA. Double-immunofluorescent staining for HA-Cog1 and β-1,4 galactosyltransferase I in patient fibroblasts transiently transfected with an expression vector encoding Cog1 with an HA tag at the C terminus for two independent transfections. (Upper) Intermediate expression of the HA-Cog1; (Lower) high overexpression. All cells overexpressing HA-Cog1 (arrows) displayed an increase in immunostaining for β-1,4 galactosyltransferase.

Fig. 5.

Model of the subunit architecture of the COG complex. This subunit connectivity map is adapted from studies of Oka et al. (9) and Ungar et al. (10). This model highlights the effects of the truncated Cog1 on the observed Cog8 expression.