Isoelectric Focusing of Serum Apolipoprotein C-III as a Sensitive Screening Method for the Detection of O-glycosylation Disturbances

Abstract

Apolipoprotein C-III (ApoC-III) is a glycoprotein carrying the most common O-linked glycan structure and is abundantly present in serum, what renders it a suitable marker for analysis of O-glycosylation abnormalities. Isoelectric focusing followed by a Western blot of ApoC-III, using PhastSystem™ Electrophoresis System (GE Healthcare), was introduced as a rather simple and rapid method for screening of certain subtypes of inherited glycosylation disorders. The study's aim was to establish this method in our laboratory, what included performing the analysis in a group of 170 healthy individuals to set the reference range of detected relative amounts of sialylated ApoC-III isoforms and to evaluate the gender- and age-dependent differences. A significant relative increase of asialo-ApoC-III with growing age was found. Secondly, we examined serum from patients with selected metabolic disorders and detected minor O-glycosylation changes in diseases such as Prader-Willi syndrome, PGM1 (phosphoglucomutase 1) or MAN1B (class 1B alpha-1,2-mannosidase) deficiency. Our results show that this method allows for a sensitive detection of ApoC-III O-glycosylation status, however this might be modulated by several factors (i.e. nutrition, medication) whose exact role remains to be determined.

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

“…In healthy adults, MALDI‐MS revealed, in terms of relative peak areas, increased levels of apoC‐III 0a (aglycosylated species) and decrease of apoC‐III 2 when compared with pediatric samples (our data; not shown). These findings are in agreement with a recent paper reporting age‐dependent apoC‐III sialylation by IEF . Therefore, we compared the results of apoC‐III glycosylation analysis of our COG5‐CDG patients with those of age‐matched healthy pediatric controls.…”

“…These findings are in agreement with a recent paper reporting age-dependent apoC-III sialylation by IEF. [23] Therefore, we compared the results of apoC-III glycosylation analysis of our COG5-CDG patients with those of age-matched healthy pediatric controls. Figure 1 shows representative MALDI mass spectra of apoC-III glycosylation profiles of a control and a patient, respectively.…”

Apolipoprotein C‐III O‐glycoform profiling of 500 serum samples by matrix‐assisted laser desorption/ionization mass spectrometry for diagnosis of congenital disorders of glycosylation

“…The apoCIII‐0a/apoCIII‐1 ratio differed significantly between the under 1 year and older age groups ( p < 0.0001, Welch's t test) (Figure 4). This result indicated that occupancy of the glycosylation site by GalNAc was high in early infancy, an observation consistent with the report of Ondrušková et al who analyzed 170 healthy individuals (age range: 1 day to 42 years) employing isoelectric focusing and found a relative rise in apoCIII‐0 with increasing age 23 . The attachment of GalNAc is probably dependent on various factors including the enzymatic activity of transferring GalNAc to apoCIII, the supply of donor sugar‐nucleotide Uridine diphosphate (UDP)‐GalNAc, the rate of production and clearance of apoCIII protein and so on.…”

“…This result indicated that occupancy of the glycosylation site by GalNAc was high in early infancy, an observation consistent with the report of Ondrušková et al who analyzed 170 healthy individuals (age range: 1 day to 42 years) employing isoelectric focusing and found a relative rise in apoCIII-0 with increasing age. 23 The attachment of GalNAc is probably dependent on various factors including the enzymatic activity of transferring GalNAc to apoCIII, the supply of donor sugar-nucleotide Uridine diphosphate (UDP)-GalNAc, the rate of production and clearance of F I G U R E 2 Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) profiling of apoCIII glycoforms. The mucin-type core-1 glycoforms corresponding to apoCIII ions and the synthetic pathway are illustrated above the mass spectrum.…”

“…Decreased sialylation of apoCIII has been reported in various forms of CDG including a deficiency of the SLC35A1 CMP-sialic acid transporter, 30 COG 1, 2, 5, 6, 7 and 8, ATP6V0A2 and other V-ATPase subunits, and PGM1. 23 These diseases constitute the group with combined N-and O-glycosylation abnormalities. The present study adds TRAPPC11-CDG and B4GALT1-CDG to this list.…”