Glycosylation Analysis for Congenital Disorders of Glycosylation

Abstract

Congenital disorders of glycosylation (CDG) are a group of diseases with highly variable phenotypes and inconsistent clinical features. Since the first description of a CDG in 1980, approximately 100 disorders have been identified. Most of these are defects in protein glycosylation, although an increasing number are defects of glycolipid or proteoglycan biosynthesis. A group of biochemical markers has been used to characterize protein glycosylation abnormalities in CDG. This unit describes three protocols that can be used to measure plasma or serum carbohydrate deficient transferrin (CDT) profile, N-glycan profile, and O-glycan profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) or liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS). The quantification of particular biomarkers, such as T antigens or sialylated T antigens, could also be achieved by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These techniques can be used to identify a majority of patients with defects in protein glycosylation, although different techniques, such as flow cytometry with immunostaining, are necessary to detect defects in glycolipid or proteoglycan biosynthesis which is not included in this unit.

The schizophrenia risk locus in SLC39A8 alters brain metal transport and plasma glycosylation

“…A2G1S1 is commonly increased in CDGs and suggestive of decreased β(1,4)galactosyltransferase activity (60). We observed a ~20% increased abundance of A2G1S1 in both CT and TT carriers, though it did not reach statistical significance.…”

“…Table 7). Two of the most abundant large N-glycans, A3G3S3 and A3FG3S3 (m/z 3603 and 3777, respectively) are consistently reduced in multiple CDGs (34). Both subject A and subject B have decreased A3G3S3 and subject A has lower A3FG3S3 at baseline; following Mn supplementation both A3G3S3 and A3FG3S3 increase in both individuals (A3G3S3: A 3.25% -> 8.03% with Mn; B 2.24% -> 5.71% with Mn; CC 3.68%), (A3FG3S3: A 0.662% -> 0.725% with Mn; B 3.81% -> 6.72% with Mn; CC 1.58%) (Supp.…”

“…N-glycans of plasma proteins were measured from 5 µL samples following peptide:N-glycosidase F (PNGaseF) cleavage and permethylation, and analyzed using MALDI-TOF MS based on standard protocols (34,35). Plasma was studied in lieu of serum given the abundance of literature on human plasma glycosylation (36), and because our pilot analysis of serum and plasma from the same donors found no substantial differences (data not shown).…”

“…The clinical phenotypes are dramatic and overlap in intellectual disability, seizures, brain structural abnormalities, low to undetectable Mn, and impaired transferrin glycosylation. Transferrin Nglycosylation is a common screening test for CDGs, though recent efforts have focused on performing mass spectrometry (MS) based methods given the more complete and sensitive nature of the test (34).…”

“…Relative abundance of A2G1S1, a monosialo-monogalacto bi-antennary N-glycan with permethylated m/z of 2227, is consistently elevated in plasma/serum across multiple CDGs (34), and was the only N-glycan reported as significantly elevated by Rader and colleagues in A391T homozygotes (28). Both subject A and subject B show increased A2G1S1 at baseline that decreased following Mn treatment (A 2.246% -> 0.652% with Mn; B 1.650% -> 0.622% with Mn; CC 0.439%) (Supp.…”