MALDI‐MS profiling of serum O‐glycosylation and N‐glycosylation in COG5‐CDG

Abstract

Congenital disorders of glycosylation (CDG) are due to defective glycosylation of glycoconjugates. Conserved oligomeric Golgi (COG)-CDG are genetic diseases due to defects of the COG complex subunits 1-8 causing N-glycan and O-glycan processing abnormalities. In COG-CDG, isoelectric focusing separation of undersialylated glycoforms of serum transferrin and apolipoprotein C-III (apoC-III) allows to detect N-glycosylation and O-glycosylation defects, respectively. COG5-CDG (COG5 subunit deficiency) is a multisystem disease with dysmorphic features, intellectual disability of variable degree, seizures, acquired microcephaly, sensory defects and autistic behavior. We applied matrix-assisted laser desorption/ionization-MS for a high-throughput screening of differential serum O-glycoform and N-glycoform in five patients with COG5-CDG. When compared with age-matched controls, COG5-CDG showed a significant increase of apoC-III (aglycosylated glycoform), whereas apoC-III (mono-sialylated glycoform) decreased significantly. Serum N-glycome of COG5-CDG patients was characterized by the relative abundance of undersialylated and undergalactosylated biantennary and triantennary glycans as well as slight increase of high-mannose structures and hybrid glycans. Using advanced and well-established MS-based approaches, the present findings reveal novel aspects on O-glycan and N-glycan profiling in COG5-CDG patients, thus providing an increase of current knowledge on glycosylation defects caused by impairment of COG subunits, in support of clinical diagnosis. Copyright © 2017 John Wiley & Sons, Ltd.

Maintaining order: COG complex controls Golgi trafficking, processing, and sorting

“…N-glycan mass spectrometry (MS) analysis confirmed hyposialylation of N-glycans, similar to CHO-COG mutants and COG-CDG patients (discussed in the next section). Fly COG mutants also displayed increased high-mannose, paucimannose, and Humans, COG4-CDG COG4 (R729W), COG4 (G516R) Cells: reduction in COG3 (50%), COG2 (40%), COG1 (25%), and COG5 (40%) protein levels, COG complex formation seemed to be unaffected, mild Golgi dysfunction (compared to COG7 or COG8-CDG), Golgi dilatation and fragmentationPatients: Saul-Wilson syndrome, a rare form of primordial dwarfism with characteristic facial and radiographic features [102,115] Humans, COG5-CDG COG5 (homozygous intronic substitution (c.1669-15T>C) leading to exon skipping) Cells: undersialylation of N-and O-glycansPatients: moderate psychomotor retardation with language delay, truncal ataxia and slight hypotonia [110,166,174,175] Humans, COG6-CDG COG6 (G549V) Cells: reduction in STX6 levels, glycosylation defects including reduced sialyation of O-glycans; decreased activity of B4GALT1 but normal import of UDP-galactose into the Golgi, reduced protein levels of COG5 (55%), COG6 (21%), and COG7 (62%), degradation of mRNA encoding COG6, formation of the COG complex affectedPatients: microcephaly, chronic inflammatory bowel disease, micronodular liver cirrhosis, severe neurologic disease characterized by vitamin K deficiency, vomiting, intractable focal seizures, intracranial bleedings and fatal outcome in early infancy [176][177][178][179] Humans, COG7-CDG COG7 (intronic splice site mutation (c.169+4A>C)) Cells: disruption of multiple N-and O-glycosylation pathways, completely destabilized COG complexPatients: growth retardation, microcephaly, hypotonia, adducted thumbs, feeding problems, failure to thrive, cardiac anomalies, wrinkled skin and episodes of extreme hyperthermia, skeletal anomalies and a mild liver involvement [96,101,173,180] Humans, COG8-CDG COG8 Cells: deficient in sialylation of both N-and O-glycans, slower brefeldin A induced disruption of the Golgi matrix, reduction in COG1, COG5, COG6, and COG7 protein levels but not COG2, COG3 and COG4, COG5, COG6, and COG7 were also mislocalizedPatients: cerebellar atrophy, Elevated blood creatine phosphokinase, Alternating esotropia, psychomotor retardation, failure to thrive, intolerance to wheat and dairy products, lack of bowel or bladder control, dry skin with keratosis pilaris, mild contractures of the lower extremities [99,100,103,107,108] Humans, TMED6-COG8 translocation…”

Sugary Logistics Gone Wrong: Membrane Trafficking and Congenital Disorders of Glycosylation

“…Clinically, patients of COG-CDGs present with prominent incomplete galactosylation and sialylation [149,153], and this is true for variants in both lobe A and B subunits. While the variants mostly consist of missense mutations or truncations for lobe A subunits and full loss-of-function mutations in lobe B subunits, glycan profiles of patients show the same hypogalactosylation and hyposialylation for both types of mutations [154,155]. This raises the question of whether mutations in one COG subunit affect the entire COG complex.…”

Clinical glycomics for the diagnosis of congenital disorders of glycosylation

“…Apo C-III has only a single O-glycan on Thr-74 which is terminally modified by up to two sialic acids to generate three main IEF isoforms: Apo C-III 0 (no sialic acid), Apo C-III 1 (one sialic acid), and Apo C-III 2 (two sialic acids). Decreased sialylation on Apo C-III profiles has been reported in conserved oligomeric Golgi (COG) defects (Spaapen et al 2005 ; Foulquier et al 2006 ; Foulquier et al 2007 ; Kranz et al 2007 ; Morava et al 2007 ; Wopereis et al 2007 ; Zeevaert et al 2008 ; Ng et al 2011 ; Palmigiano et al 2017 ) and autosomal recessive cutis laxa type-2 (ARCL2) due to ATP6V0A2 dysfunctions (Morava et al 2005 ; Kornak et al 2008 ). The limitation of Apo C-III IEF is that it is not able to differentiate between the three possible Apo C-III 0 isoforms; the “real unglycosylated Apo C-III” and Apo C-III with two non-sialylated monosaccharides namely Gal and GalNAc.…”