生物化学

Dolichol diphosphate-linked oligosaccharides (LLO) are the sugar donors in N-glycosylation, a fundamental protein post-translational modification of the eukaryotic secretory pathway. Defects in LLO biosynthesis produce human Congenital Disorders of Glycosylation Type I. The synthesis of LLOs and the transfer reactions to their protein acceptors is highly conserved among animal, plant, and fungi kingdoms, making the fission yeast Schizosaccharomyces pombe a suitable model to study these processes. Here, we present a protocol to determine the LLO patterns produced in vivo by S. pombe cells that may be easily adapted to other cell types. First, exponentially growing cultures are labeled with a pulse of [¹⁴C]-glucose. LLOs are then purified by successive extractions with organic solvents, and glycans are separated from the lipid moieties in mild acid hydrolysis and a new solvent extraction. The purified glycans are then run on paper chromatography. We use a deconvolution process to adjust the profile obtained to the minimal number of Gaussian functions needed to fit the data and determine the proportion of each species with respect to total glycan species present in the cell. The method we provide here might be used without any expensive or specialized equipment. The deconvolution process described here might also be useful to analyze species in non-completely resolved chromatograms.

Graphical abstract:

Workflow for the labeling, extraction, separation, and identification of LLO species in S. pombe.

(A) Radioactive pulse of S. pombe cells with [¹⁴C]-glucose for 15 min at 28 °C. (B) Organic extraction of LLOs from labeled yeasts sequentially using methanol, chloroform, H₂O, chloroform:methanol:H₂O (1:1:0.3), 0.02 M HCl (to separate glycans from dolichol), and chloroform:methanol:H₂O (1:16:16). (C) Preparation of the sample for chromatography on paper: drying by airflow and radioactivity check. (D) Loading of samples in chromatographic paper and descendent chromatography in a glass chamber. The obtained plots (CPM versus running distance) need to be analyzed to identify single glycan species.

Heparanase, an endo-β-D-glucuronidase, cleaves cell surface and extracellular matrix heparan sulfate (HS) chains at distinct sites and plays important biological roles including modulation of cell growth and metastasis. Although a number of different types of heparanase assays have been reported to date, most are labor intensive, complex and/or expensive to carry out. We reasoned that a simpler heparanase assay could be developed using heparin labeled with Dabcyl and EDANS as donor and acceptor fluorophores so as to generate a FRET signal. Our results show that a more robust heparanase assay could be developed based on the principle studied herein and more homogeneous preparation of heparin. Yet, the assay in its current form could be used for routine screening of potential inhibitors in a high-throughput manner as well as for studying heparanase activity expressed in tumors as well as biological fluids like plasma.

Glycosylation of asparagine residues is widespread in Eukarya, and occurs in virtually all Archaea and some eubacterial species. A membrane-bound enzyme, oligosaccharyltransferase, catalyzes the transfer of an oligosaccharide chain from a sugar donor (lipid-linked oligosaccharide, LLO) to an asparagine residue in the consensus sequence, Asn-X-Ser/Thr (X ≠ Pro), in proteins. The in vitro oligosaccharyl transfer assay reaction mixture contains a detergent-solubilized oligosaccharyltransferase (OST), a sugar donor LLO, and a sugar acceptor peptide. Previous assay methods are problematic, in terms of the use of radioactive compounds and the cumbersome separation procedures using lectin binding or two-phase partitioning. Here, we describe a new oligosaccharyl transfer assay method, which is radioisotope-free and relies on a different separation mechanism. The glycopeptide products are separated from unreacted peptides by SDS-PAGE. A fluorescent dye is attached to the peptide substrate during custom peptide synthesis. The fluorescent imaging of the SDS-PAGE gels ensures high sensitivity and quantitative performance. The user-friendly PAGE format is particularly suitable for presentation in scientific papers. For illustrative applications, time-course and peptide library experiments are shown.

Modifications of N-linked oligosaccharides of glycoproteins soon after their biosynthesis correlate to glycoprotein folding status. These alterations can be detected in a sensitive way by pulse-chase analysis of [2-³H]mannose-labeled glycoproteins, with enzymatic removal of labeled N-glycans, separation according to size by HPLC and radioactive detection in a scintillation counter.