Comprehensive Profiling of Glycosphingolipid Glycans Using a Novel Broad Specificity Endoglycoceramidase in a High-Throughput Workflow

Abstract

The biological function of glycosphingolipids (GSLs) is largely determined by their glycan headgroup moiety. This has placed a renewed emphasis on detailed GSL headgroup structural analysis. Comprehensive profiling of GSL headgroups in biological samples requires the use of endoglycoceramidases with broad substrate specificity and a robust workflow that enables their high-throughput analysis. We present here the first high-throughput glyco-analytical platform for GSL headgroup profiling. The workflow features enzymatic release of GSL glycans with a novel broad-specificity endoglycoceramidase I (EGCase I) from Rhodococcus triatomea, selective glycan capture on hydrazide beads on a robotics platform, 2AB-fluorescent glycan labeling, and analysis by UPLC-HILIC-FLD. R. triatomea EGCase I displayed a wider specificity than known EGCases and was able to efficiently hydrolyze gangliosides, globosides, (n)Lc-type GSLs, and cerebrosides. Our workflow was validated on purified GSL standard lipids and was applied to the characterization of GSLs extracted from several mammalian cell lines and human serum. This study should facilitate the analytical workflow in functional glycomics studies and biomarker discovery.

Persistent reduction in sialylation of cerebral glycoproteins following postnatal inflammatory exposure

“…Recombinant EGCase I tagged with N-terminal His tag has been expressed in E. coli and purified using Ni-NTA superflow column as described [ 34 ]. The activity of purified EGCase I was tested using GM3 as a substrate.…”

Intact glycosphingolipidomic analysis of the cell membrane during differentiation yields extensive glycan and lipid changes

“…However, these approaches are applicable to only a few glycan epitopes for which there are known lectins, and they are unable to reveal the nature of the ceramide. More recent efforts make use of enzymatic digestion to cleave the lipid portion from the glycan headgroups before analyzing the portions separately, at the expense of information about how these two portions are connected 21 , 22 .…”

Modern separation techniques coupled to high performance mass spectrometry for glycolipid analysis

“…In glycolipidomics UPLC‐HILIC MS was developed for high‐throughput profiling of GLs glycans released from several mammalian cell lines and human serum by endoglycoceramidase I (EGCase I) from Rhodococcus triatomea . The first step of this method involves the digestion of GLs through their suspension in sodium acetate buffer (pH 5.2) containing 1 mg/mL Triton‐X‐100 and incubation at 37°C for 16 h using different concentrations (2−120 mU) of R. triatomea rEGCase I or R. equi 2 mU of rEGCase II in a total volume of 20−200 μL.…”

“…The first step of this method involves the digestion of GLs through their suspension in sodium acetate buffer (pH 5.2) containing 1 mg/mL Triton‐X‐100 and incubation at 37°C for 16 h using different concentrations (2−120 mU) of R. triatomea rEGCase I or R. equi 2 mU of rEGCase II in a total volume of 20−200 μL. The released glycan head groups, captured and cleaned using a Hamilton Robotics StarLet liquid handling platform and fluorescent labeled with 2‐aminobenzamide were further analyzed by UPLC‐HILIC with fluorescence detection MS .…”