Background

N-glycosylation is a frequent post-translational modification in the secretory pathway of eukaryotic cells involved in glycoprotein folding in the endoplasmic reticulum (ER). During N-glycosylation, a glycan composed of three glucoses (Glc), nine mannoses (Man), and two N-acetyl glucosamines (G3M9) is transferred in a single reaction step from a dolichol diphosphate oligosaccharide (LLO) donor to a consensus sequence present in proteins entering the ER (Cali et al., 2008). The reaction is catalyzed by the oligosaccharyltransferase (OST), which discriminates among completed and non-completed LLOs. The LLOs are synthesized stepwise in the ER membrane by different ALG gene products, first in the cytosolic side and then in the luminal side of the ER membrane (Figure 1) (Aebi, 2013; Stanley et al., 2015). Defects in LLOs synthesis result in their poor recognition by OST and thus less efficient transfer to proteins, leaving empty some normally occupied N-glycosylation sites. This hypoglycosylation of proteins may have profound effects in the cell and lead to several different Congenital Disorders of Glycosylation (CDG) type I (Ondruskova et al., 2021). These diseases are difficult to diagnose. The first confirmation usually arrives when hypoglycosylation is observed in serum transferrin, and a defect in LLO synthesis is assumed. To know which ALG genes are defective in each patient, either the biochemical analysis of LLO or a whole exome sequencing are required (Chang et al., 2018; Ng and Freeze, 2018; Wilson and Matthijs, 2021). The core machinery of LLO synthesis and N-glycosylation is highly conserved among eukaryotic species (Aebi, 2013), particularly between the fission yeast Schizosaccharomyces pombe and mammals (Fernandez et al., 1994). S. pombe is easily handled, facilities required for its growth and maintenance are inexpensive, and many biochemical and genetic tools are already developed for this organism. For these reasons, S. pombe has become a suitable model organism for research, and several molecular tools developed and optimized for use in this model organism have been used in mammalian cells (Hagan, 2016).

We present here a detailed protocol to specifically identify the LLO species synthesized in S. pombe in vivo. The key step of the protocol is to stop protein synthesis with inhibitors preventing glycan transfer to proteins while radioactive labeled LLOs are being synthesized. Cultures are compared under similar nutritional growth conditions, in order to avoid differences in LLO structures due to environmental conditions or metabolic constraints (Gershman and Robbins, 1981; Chapman and Calhoun, 1988). We then extract LLOs from the labeled cells and analyze them either by HPLC or by paper chromatography. To better identify the peaks obtained, we developed a deconvolution process using a Gaussian function available in a Microsoft Excel Solver datasheet provided here. This method allows a better visualization of the peaks, making them easily comparable with the standards run in parallel, and provides an accurate quantification of each peak. With the protocol described here, we were able to distinguish the LLOs produced in 16 S. pombe strains that synthesize combinatorial possible structures of LLOs (GXMY, where X is the number of Glc residues in the glycan, ranging from three to zero, and Y is the number of Man units, ranging from nine to five) (Gallo et al., 2022). This low-cost method has the advantages of being readily available for almost all laboratories, providing high accuracy, and being easily adapted to any cell type, simplifying the determination of the LLO biosynthetic step blocked in a particular cell mutant or synthesized by the organism being studied. Moreover, the method we provide to obtain the deconvolution of the LLO profiles may be used for other chromatographic resolutions.

Figure 1. Synthesis of LLOs and glycan transfer to nascent proteins in the ER. The G3M9 LLO is built in the ER membrane step-by-step by monosaccharide addition mediated by specific glycosyltransferases encoded by alg (asparagine-linked glycosylation) genes. Two N-acetylglucosamines and five Man are transferred to the dolichol from nucleotide-sugars on the cytoplasmic side of the ER membrane. Then, the LLO intermediate bearing five Man residues is flipped to the ER lumen, where four additional Man residues are added by the ALG3-, ALG9-, and ALG12-encoded ER luminal dolichol-dependent mannosyltransferases. Finally, three Glc residues are incorporated by the ALG6-, ALG8-, and ALG10-encoded dolichol-dependent glucosyltransferases. Once completed, the LLO is transferred by OST to nascent polypeptides entering the ER through the translocon.