Shiteshu Shrimal

Serum-dependent selective expression of EhTMKB1-9, a member of Entamoeba histolytica B1 family of transmembrane kinases.

Entamoeba histolytica transmembrane kinases (EhTMKs) can be grouped into six distinct families on the basis of motifs and sequences. Analysis of the E. histolytica genome revealed the presence of 35 EhTMKB1 members on the basis of sequence identity (≥95%). Only six homologs were full length containing an extracellular domain, a transmembrane segment and an intracellular kinase domain. Reverse transcription followed by polymerase chain reaction (RT-PCR) of the kinase domain was used to generate a library of expressed sequences. Sequencing of randomly picked clones from this library revealed that about 95% of the clones were identical with a single member, EhTMKB1-9, in proliferating cells. On serum starvation, the relative number of EhTMKB1-9 derived sequences decreased with concomitant increase in the sequences derived from another member, EhTMKB1-18. The change in their relative expression was quantified by real time PCR. Northern analysis and RNase protection assay were used to study the temporal nature of EhTMKB1-9 expression after serum replenishment of starved cells. The results showed that the expression of EhTMKB1-9 was sinusoidal. Specific transcriptional induction of EhTMKB1-9 upon serum replenishment was further confirmed by reporter gene (luciferase) expression and the upstream sequence responsible for serum responsiveness was identified. EhTMKB1-9 is one of the first examples of an inducible gene in Entamoeba. The protein encoded by this member was functionally characterized. The recombinant kinase domain of EhTMKB1-9 displayed protein kinase activity. It is likely to have dual specificity as judged from its sensitivity to different kinase inhibitors. Immuno-localization showed EhTMKB1-9 to be a surface protein which decreased on serum starvation and got relocalized on serum replenishment. Cell lines expressing either EhTMKB1-9 without kinase domain, or EhTMKB1-9 antisense RNA, showed decreased cellular proliferation and target cell killing. Our results suggest that E. histolytica TMKs of B1 family are functional kinases likely to be involved in serum response and cellular proliferation.

Cotranslational and posttranslocational N-glycosylation of proteins in the endoplasmic reticulum.

Asparagine linked glycosylation of proteins is an essential protein modification reaction in most eukaryotic organisms. N-linked oligosaccharides are important for protein folding and stability, biosynthetic quality control, intracellular traffic and the physiological function of many N-glycosylated proteins. In metazoan organisms, the oligosaccharyltransferase is composed of a catalytic subunit (STT3A or STT3B) and a set of accessory subunits. Duplication of the catalytic subunit gene allowed cells to evolve OST complexes that act sequentially to maximize the glycosylation efficiency of the large number of proteins that are glycosylated in metazoan organisms. We will summarize recent progress in understanding the mechanism of (a) cotranslational glycosylation by the translocation channel associated STT3A complex, (b) the role of the STT3B complex in mediating cotranslational or posttranslocational glycosylation of acceptor sites that have been skipped by the STT3A complex, and (c) the role of the oxidoreductase MagT1 in STT3B-dependent glycosylation of cysteine-proximal acceptor sites.

Extreme C-terminal sites are posttranslocationally glycosylated by the STT3B isoform of the OST

Glycosylation in the C-terminal 50–55 residues of proteins is mediated posttranslocationally by the STT3B isoform of oligosaccharyltransferase, with a preference for NXT sites.

Mutations in STT3A and STT3B cause two congenital disorders of glycosylation

We describe two unreported types of congenital disorders of glycosylation (CDG) which are caused by mutations in different isoforms of the catalytic subunit of the oligosaccharyltransferase (OST). Each isoform is encoded by a different gene (STT3A or STT3B), resides in a different OST complex and has distinct donor and acceptor substrate specificities with partially overlapping functions in N-glycosylation. The two cases from unrelated consanguineous families both show neurologic abnormalities, hypotonia, intellectual disability, failure to thrive and feeding problems. A homozygous mutation (c.1877T > C) in STT3A causes a p.Val626Ala change and a homozygous intronic mutation (c.1539 + 20G > T) in STT3B causes the other disorder. Both mutations impair glycosylation of a GFP biomarker and are rescued with the corresponding cDNA. Glycosylation of STT3A- and STT3B-specific acceptors is decreased in fibroblasts carrying the corresponding mutated gene and expression of the STT3A (p.Val626Ala) allele in STT3A-deficient HeLa cells does not rescue glycosylation. No additional cases were found in our collection or in reviewing various databases. The STT3A mutation significantly impairs glycosylation of the biomarker transferrin, but the STT3B mutation only slightly affects its glycosylation. Additional cases of STT3B-CDG may be missed by transferrin analysis and will require exome or genome sequencing.

N-linked glycosylation and homeostasis of the endoplasmic reticulum

As a major site of protein biosynthesis, homeostasis of the endoplasmic reticulum is critical for cell viability. Asparagine linked glycosylation of newly synthesized proteins by the oligosaccharyltransferase plays a central role in ER homeostasis due to the use of protein-linked oligosaccharides as recognition and timing markers for glycoprotein quality control pathways that discriminate between correctly folded proteins and terminally malfolded proteins destined for ER associated degradation. Recent findings indicate how the oligosaccharyltransferase achieves efficient and accurate glycosylation of the diverse proteins that enter the endoplasmic reticulum. In metazoan organisms two distinct OST complexes cooperate to maximize the glycosylation of nascent proteins. The STT3B complex glycosylates acceptor sites that have been skipped by the translocation channel associated STT3A complex.

Oxidoreductase activity is necessary for N-glycosylation of cysteine-proximal acceptor sites in glycoproteins

The oxidoreductases activity of MagT1, an ER-localized thioredoxin homologue, is necessary for posttranslocational N-glycosylation of acceptor sites that lie proximal to cysteine residues.

Glycosylation of closely spaced acceptor sites in human glycoproteins

Summary Asparagine-linked glycosylation of proteins by the oligosaccharyltransferase (OST) occurs when acceptor sites or sequons (N-x≠P-T/S) on nascent polypeptides enter the lumen of the rough endoplasmic reticulum. Metazoan organisms assemble two isoforms of the OST that have different catalytic subunits (STT3A or STT3B) and partially non-overlapping cellular roles. Potential glycosylation sites move past the STT3A complex, which is associated with the translocation channel, at the protein synthesis elongation rate. Here, we investigated whether close spacing between acceptor sites in a nascent protein promotes site skipping by the STT3A complex. Biosynthetic analysis of four human glycoproteins revealed that closely spaced sites are efficiently glycosylated by an STT3B-independent process unless the sequons contain non-optimal sequence features, including extreme close spacing between sequons (e.g. NxTNxT) or the presence of paired NxS sequons (e.g. NxSANxS). Many, but not all, glycosylation sites that are skipped by the STT3A complex can be glycosylated by the STT3B complex. Analysis of a murine glycoprotein database revealed that closely spaced sequons are surprisingly common, and are enriched for paired NxT sites when the gap between sequons is less than three residues.

Oligosaccharyltransferase inhibition induces senescence in RTK-driven tumor cells

Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high-throughput screen and lead-compound-optimization campaign that delivered a cell-permeable inhibitor, NGI-1. NGI-1 targets oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.

Reduced expression of the oligosaccharyltransferase exacerbates protein hypoglycosylation in cells lacking the fully assembled oligosaccharide donor

A defect in the assembly of the oligosaccharide donor (Dol-PP-GlcNAc(2)Man(9)Glc(3)) for N-linked glycosylation causes hypoglycosylation of proteins by the oligosaccharyltransferase (OST). Mammalian cells express two OST complexes that have different catalytic subunits (STT3A or STT3B). We monitored glycosylation of proteins in asparagine-linked glycosylation 6 (ALG6) deficient cell lines that assemble Dol-PP-GlcNAc(2)Man(9) as the largest oligosaccharide donor. Based upon pulse labeling experiments, 30-40% of STT3A-dependent glycosylation sites and 20% of STT3B-dependent sites are skipped in ALG6-congenital disorders of glycosylation fibroblasts supporting previous evidence that the STT3B complex has a relaxed preference for the fully assembled oligosaccharide donor. Glycosylation of STT3B-dependent sites was more severely reduced in the ALG6 deficient MI8-5 cell line. Protein immunoblot analysis and RT-PCR revealed that MI8-5 cells express 2-fold lower levels of STT3B than the parental Chinese hamster ovary cells. The combination of reduced expression of STT3B and the lack of the optimal Dol-PP-GlcNAc(2)Man(9)Glc(3) donor synergize to cause very severe hypoglycosylation of proteins in MI8-5 cells. Thus, differences in OST subunit expression can modify the severity of hypoglycosylation displayed by cells with a primary defect in the dolichol oligosaccharide assembly pathway.

Identification and Partial Characterization of a Dynamin-Like Protein, EhDLP1, from the Protist Parasite Entamoeba histolytica

ABSTRACT The dynamin superfamily of proteins includes a large repertoire of evolutionarily conserved GTPases that interact with different subcellular organelle membranes in eukaryotes. Dynamins are thought to participate in a number of cellular processes involving membrane remodeling and scission. Dynamin-like proteins (DLPs) form a subfamily of this vast class and play important roles in cellular processes, such as mitochondrial fission, cytokinesis, and endocytosis. In the present study, a gene encoding a dynamin-like protein (EhDLP1) from the protist parasite Entamoeba histolytica was identified and the protein was partially characterized using a combination of in silico, biochemical, and imaging methods. The protein was capable of GTP binding and hydrolysis, lipid binding, and oligomerization. Immunofluorescence studies showed the protein to be associated with the nuclear membrane. A mutant of EhDLP1 lacking GTP binding and hydrolyzing activities did not associate with the nuclear membrane. The results suggest a nucleus-associated function for EhDLP1.