ALG3-CDG is a rare congenital disorder of glycosylation (CDG) with a clinical phenotype that includes neurological manifestations, transaminitis, and frequent infections. The ALG3 enzyme catalyzes the first step of endoplasmic reticulum (ER) luminal glycan extension by adding mannose from Dol-P-Man to Dol-PP-Man5GlcNAc2 (Man5) forming Dol-PP-Man6. Such glycan extension is the first and fastest cellular response to ER stress, which is deficient in ALG3-CDG. In this study, we provide evidence that the unfolded protein response (UPR) and ER-associated degradation activities are increased in ALG3-CDG patient-derived cultured skin fibroblasts and there is constitutive activation of UPR mediated by the IRE1-α pathway. In addition, we show that N-linked Man3-4 glycans are increased in cellular glycoproteins and secreted plasma glycoproteins with hepatic or non-hepatic origin. We found that like other CDGs such as ALG1- or PMM2-CDG, in transferrin, the assembling intermediate Man5 in ALG3-CDG, are likely further processed into a distinct glycan, NeuAc1Gal1GlcNAc1Man3GlcNAc2, probably by Golgi mannosidases and glycosyltransferases. We predict it to be a mono-antennary glycan with the same molecular weight as the truncated glycan described in MGAT2-CDG. In summary, this study elucidates multiple previously unrecognized biochemical consequences of the glycan extension deficiency in ALG3-CDG which will have important implications in the pathogenesis of CDG.

In humans, almost all the cell surface and secreted glycoproteins are modified with complex-type N-glycans. Thus, it is essential to obtain complex-type N-glycans to fully understand the biological properties of glycoproteins. Here, human β-1,2-N-acetylglucosaminyltransferase II (hGnT-II), a Golgi-localized enzyme integral to complex-type N-glycan biosynthesis, was cloned as a truncated transmembrane form (GnT-II-ΔTM) and heterologously overexpressed in Escherichia coli. Our results showed that hGnT-II could be overexpressed in its soluble form by fusing the truncated enzyme with a thioredoxin (Trx)-tag in the Rosetta-Gami 2 strain. Using the optimized induction conditions, the expression level of recombinant protein was enhanced to yield approximately 4 mg per liter culture after affinity purification. The enzyme exhibited appropriate glycosyltransferase activity, and the calculated Km value was 52.4 μM, similar to the protein expressed in mammalian cells. Furthermore, the effect of MGAT2-CDG mutations on enzyme activity was also measured. These results suggested that the E. coli expression system was capable of the large-scale production of bioactive hGnT-II, which can be used for functional study and effective synthesis of complex-type N-glycans.

Glycosylation is a critical post/peri-translational modification required for the appropriate development and function of the immune system. As an example, abnormalities in glycosylation can cause antibody deficiency and reduced lymphocyte signaling, although the phenotype can be complex given the diverse roles of glycosylation. Human MGAT2 encodes N-acetylglucosaminyltransferase II, which is a critical enzyme in the processing of oligomannose to complex N-glycans. Complex N-glycans are essential for immune system functionality, but only one individual with MGAT2-CDG has been described to have an abnormal immunologic evaluation. MGAT2-CDG (CDG-IIa) is a congenital disorder of glycosylation (CDG) associated with profound global developmental disability, hypotonia, early onset epilepsy, and other multisystem manifestations. Here, we report a 4-year old female with MGAT2-CDG due to a novel homozygous pathogenic variant in MGAT2, a 4-base pair deletion, c.1006_1009delGACA. In addition to clinical features previously described in MGAT2-CDG, she experienced episodic asystole, persistent hypogammaglobulinemia, and defective ex vivo mitogen and antigen proliferative responses, but intact specific vaccine antibody titers. Her infection history has been mild despite the testing abnormalities. We compare this patient to the 15 previously reported patients in the literature, thus expanding both the genotypic and phenotypic spectrum for MGAT2-CDG.

Congenital disorders of glycosylation (CDG) are rare autosomal genetic diseases affecting the glycosylation of proteins and lipids. Since CDG-related clinical symptoms are classically extremely variable and nonspecific, a combination of electrophoretic, mass spectrometric, and gene sequencing techniques is often mandatory for obtaining a definitive CDG diagnosis, as well as identifying causative gene mutations and deciphering the underlying biochemical mechanisms. Here, we illustrate the potential of integrating data from capillary electrophoresis of transferrin, two-dimensional electrophoresis of N- and O-glycoproteins, mass spectrometry analyses of total serum N-linked glycans and mucin core1 O-glycosylated apolipoprotein C-III for the determination of various culprit CDG gene mutations. "Step-by-step" diagnosis pathways of four particular and new CDG cases, including MGAT2-CDG, ATP6V0A2-CDG, SLC35A2-CDG, and SLC35A3-CDG, are described as illustrative examples.

We retrospectively reviewed Saudi patients who had a congenital disorder of glycosylation (CDG). Twenty-seven Saudi patients (14 males, 13 females) from 13 unrelated families were identified. Based on molecular studies, the 27 CDG patients were classified into different subtypes: ALG9-CDG (8 patients, 29.5%), ALG3-CDG (7 patients, 26%), COG6-CDG (7 patients, 26%), MGAT2-CDG (3 patients, 11%), SLC35A2-CDG (1 patient), and PMM2-CDG (1 patient). All the patients had homozygous gene mutations. The combined carrier frequency of CDG for the encountered founder mutations in the Saudi population is 11.5 per 10,000, which translates to a minimum disease burden of 14 patients per 1,000,000. Our study provides comprehensive epidemiologic information and prevalence figures for each of these CDG in a large cohort of congenital disorder of glycosylation patients.