Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum

N-glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteins and N-glycans in microalgae. Here, N-glycoproteomic and N-glycomic approaches were used to unveil the N-glycoproteins and N-glycans in the model diatom Phaeodactylum tricornutum. In total, 863 different N-glycopeptides corresponding to 639 N-glycoproteins were identified from P. tricornutum. These N-glycoproteins participated in a variety of important metabolic pathways in P. tricornutum. Twelve proteins participating in the N-glycosylation pathway were identified as N-glycoproteins, indicating that the N-glycosylation of these proteins might be important for the protein N-glycosylation pathway. Subsequently, 69 N-glycans corresponding to 59 N-glycoproteins were identified and classified into high mannose and hybrid type N-glycans. High mannose type N-glycans contained four different classes, such as Man-5, Man-7, Man-9, and Man-10 with a terminal glucose residue. Hybrid type N-glycan harbored Man-4 with a terminal GlcNAc residue. The identification of N-glycosylation on nascent proteins expanded our understanding of this modification at a N-glycoproteomic scale, the analysis of N-glycan structures updated the N-glycan database in microalgae. The results obtained from this study facilitate the elucidation of the precise function of these N-glycoproteins and are beneficial for future designing the microalga to produce the functional humanized biopharmaceutical N-glycoproteins for the clinical therapeutics.

Identifying Components of a Halobacterium salinarum N-Glycosylation Pathway

Whereas N-glycosylation is a seemingly universal process in Archaea, pathways of N-glycosylation have only been experimentally verified in a mere handful of species. Toward expanding the number of delineated archaeal N-glycosylation pathways, the involvement of the putative Halobacterium salinarum glycosyltransferases VNG1067G, VNG1066C, and VNG1062G in the assembly of an N-linked tetrasaccharide decorating glycoproteins in this species was addressed. Following deletion of each encoding gene, the impact on N-glycosylation of the S-layer glycoprotein and archaellins, major glycoproteins in this organism, was assessed by mass spectrometry. Likewise, the pool of dolichol phosphate, the lipid upon which this glycan is assembled, was also considered in each deletion strain. Finally, the impacts of such deletions were characterized in a series of biochemical, structural and physiological assays. The results revealed that VNG1067G, VNG1066C, and VNG1062G, renamed Agl25, Agl26, and Agl27 according to the nomenclature used for archaeal N-glycosylation pathway components, are responsible for adding the second, third and fourth sugars of the N-linked tetrasaccharide decorating Hbt. salinarum glycoproteins. Moreover, this study demonstrated how compromised N-glycosylation affects various facets of Hbt. salinarum cell behavior, including the transcription of archaellin-encoding genes.

Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans

Therapeutic antibodies are decorated with complex-type N-glycans that significantly affect their biodistribution and bioactivity. The N-glycan structures on antibodies are incompletely processed in wild-type CHO cells due to their limited glycosylation capacity. To improve N-glycan processing, glycosyltransferase genes have been traditionally overexpressed in CHO cells to engineer the cellular N-glycosylation pathway by using random integration, which is often associated with large clonal variations in gene expression levels. In order to minimize the clonal variations, we used recombinase-mediated-cassette-exchange (RMCE) technology to overexpress a panel of 42 human glycosyltransferase genes to screen their impact on antibody N-linked glycosylation. The bottlenecks in the N-glycosylation pathway were identified and then released by overexpressing single or multiple critical genes. Overexpressing B4GalT1 gene alone in the CHO cells produced antibodies with more than 80% galactosylated bi-antennary N-glycans. Combinatorial overexpression of B4GalT1 and ST6Gal1 produced antibodies containing more than 70% sialylated bi-antennary N-glycans. In addition, antibodies with various tri-antennary N-glycans were obtained for the first time by overexpressing MGAT5 alone or in combination with B4GalT1 and ST6Gal1. The various N-glycan structures and the method for producing them in this work provide opportunities to study the glycan structure-and-function and develop novel recombinant antibodies for addressing different therapeutic applications.

Comprehensive glycoproteomics shines new light on the complexity and extent of glycosylation in archaea

Glycosylation is one of the most complex posttranslational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility, and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here, we extend the phenotypic characterization of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type (WT) and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project (ArcPP), we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provide new insights into the roles of N-glycosylation in archaeal cell biology.

Compromised N-Glycosylation Processing of Kv3.1b Correlates with Perturbed Motor Neuron Structure and Locomotor Activity

Neurological difficulties commonly accompany individuals suffering from congenital disorders of glycosylation, resulting from defects in the N-glycosylation pathway. Vacant N-glycosylation sites (N220 and N229) of Kv3, voltage-gated K+ channels of high-firing neurons, deeply perturb channel activity in neuroblastoma (NB) cells. Here we examined neuron development, localization, and activity of Kv3 channels in wildtype AB zebrafish and CRISPR/Cas9 engineered NB cells, due to perturbations in N-glycosylation processing of Kv3.1b. We showed that caudal primary (CaP) motor neurons of zebrafish spinal cord transiently expressing fully glycosylated (WT) Kv3.1b have stereotypical morphology, while CaP neurons expressing partially glycosylated (N220Q) Kv3.1b showed severe maldevelopment with incomplete axonal branching and extension around the ventral musculature. Consequently, larvae expressing N220Q in CaP neurons had impaired swimming locomotor activity. We showed that replacement of complex N-glycans with oligomannose attached to Kv3.1b and at cell surface lessened Kv3.1b dispersal to outgrowths by altering the number, size, and density of Kv3.1b-containing particles in membranes of rat neuroblastoma cells. Opening and closing rates were slowed in Kv3 channels containing Kv3.1b with oligomannose, instead of complex N-glycans, which suggested a reduction in the intrinsic dynamics of the Kv3.1b α-subunit. Thus, N-glycosylation processing of Kv3.1b regulates neuronal development and excitability, thereby controlling motor activity.

Functional analysis of Ost3p and Ost6p containing yeast oligosaccharyltransferases

The oligosaccharyltransferase (OST) is the central enzyme in the N‑glycosylation pathway. It transfers a defined oligosaccharide from a lipid-linker onto the asparagine side chain of proteins. The yeast OST consists of eight subunits and exists in two catalytically distinct isoforms that differ in one subunit, Ost3p or Ost6p. The cryo-electron microscopy structure of the Ost6p containing complex was found to be highly similar to the Ost3p containing OST. OST enzymes with altered Ost3p/Ost6p subunits were generated and functionally analysed. The three C-terminal transmembrane helices were responsible for the higher turnover-rate of the Ost3p vs. the Ost6p containing enzyme in vitro and the more severe hypoglycosylation in Ost3p lacking strains in vivo. Glycosylation of specific OST target sites required the N-terminal thioredoxin domain of Ost3p or Ost6p. This Ost3p/Ost6p dependence was glycosylation site but not protein specific. We concluded that the Ost3p/Ost6p subunits modulate the catalytic activity of OST and provide additional specificity for OST substrate recognition.

Nuclear receptors FXR and SHP regulate protein N-glycan modifications in the liver

Nuclear receptors farnesoid X receptor (FXR) and small heterodimer partner (SHP) are key regulators of metabolism. Here, we report a previously unknown function for the hepatic FXR-SHP axis in controlling protein N-linked glycosylation. Transcriptome analysis in liver-specific Fxr-Shp double knockout (LDKO) livers revealed induction of genes encoding enzymes in the N-glycosylation pathway, including Mgat5, Fut8, St3gal6, and St6gal1. FXR activation suppressed Mgat5, while Shp deletion induced St3gal6 and St6gal1. Increased percentages of core-fucosylated and triantennary glycan moieties were seen in LDKO livers, and proteins with the “hyperglycoforms” preferentially localized to exosomes and lysosomes. This up-regulation of N-glycosylation machinery was specific to the Golgi apparatus and not the endoplasmic reticulum. The increased glycan complexity in the LDKO correlated well with dilated unstacked Golgi ribbons and alterations in the secretion of albumin, cholesterol, and triglycerides. Our findings demonstrate a role for the FXR-SHP axis in maintaining glycoprotein diversity in the liver.