Site-specific glycosylation in animal cells. Substitution of glutamine for asparagine 293 in chicken ovalbumin does not allow glycosylation of asparagine 312

It has been shown previously that chicken ovalbumin synthesized and secreted in a heterologous cell system is glycosylated at the correct site and that the oligosaccharides at that site, similar to the protein made in hen oviduct, are predominantly of the hybrid type (Sheares, B. T., and Robbins, P. W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1993-1997). This site-specific glycosylation of Asn293, but not Asn312, suggested a prominent role for the nascent protein chain rather than the specific cell type in directing the proper attachment of oligosaccharide chains. In the present study, the effect of glycosylation at Asn293 on the glycosylation of Asn312 has been investigated. Using a 20-base oligodeoxynucleotide primer containing a 2-base mismatch, the codon for Asn293 in the chicken ovalbumin gene (AAC) was changed to that for Gln (CAA), thereby preventing glycosylation at amino acid 293. Constructions containing this mutation were transfected into mouse L (tk-) cells which were subsequently labeled with [35S]methionine. Ovalbumin secreted by these cells was recovered by immunoaffinity chromatography and analyzed for the presence of an oligosaccharide attached at Asn312. Treatment of the material with peptide:N-glycosidase F demonstrated that ovalbumin molecules containing Gln substituted for Asn293 were not glycosylated. This further supports our earlier hypothesis that the nascent protein chain is responsible for directing site-specific glycosylation of ovalbumin, and that the presence of an oligosaccharide chain at the first site has no influence on glycosylation at the second site.     

Role of glycosylation in secretion of yeast acid phosphatase

The minimal glycosylation requirement for acid phosphatase secretion and activity was investigated using tunicamycin, an inhibitor of protein glycosylation, and a yeast mutant defective in the synthesis of oligosaccharide outer chains. The results obtained show that outer chain addition is not essential for secretion of active enzyme and that only 4 core chains, out of 8 normally attached to a protein subunit, are sufficient for enzyme transport to the periplasmic space. Enzyme forms with less than 4 chains were retained in membranes of endoplasmic reticulum. Secreted underglycosylated enzyme forms are partially or completely inactive.     

Protein glycosylation in yeast

S. cerevisiae contains many mannose-rich glycoproteins that possess N- and O-linked carbohydrate chains, and both types may even occur on one and the same protein. The steps in the synthesis of asparagine-linked chains begin with assembly and transfer of the lipid-linked precursor to protein in a way common to all eucaryotes. Subsequent modifications lead to mannosyl extensions of various lengths, but complex type carbohydrate structures are not formed. Oligosaccharides O-linked to serine/threonine consist exclusively of mannose in S. cerevisiae. The mannose residue attached directly to the protein is transferred from Dol-P-Man in a unique way, which has been observed so far for fungal cells only. The cellular localization of the glycosylation reactions is summarized and the problem of transmembrane translocation of the sugar precursors at the ER and the Golgi is discussed. Some aspects of secretory (sec) and asparagine linked glycosylation (alg) mutants have been covered, and the various hypotheses related to the possible functions of this costly protein modification process are discussed. The article may also be helpful for those, who want to exploit the yeast's protein synthesizing machinery by genetically manipulating the cells.     

Rps5-Rps16 communication is essential for efficient translation initiation in yeast S. cerevisiae

Conserved ribosomal proteins frequently harbor additional segments in eukaryotes not found in bacteria, which could facilitate eukaryotic-specific reactions in the initiation phase of protein synthesis. Here we provide evidence showing that truncation of the N-terminal domain (NTD) of yeast Rps5 (absent in bacterial ortholog S7) impairs translation initiation, cell growth and induction of GCN4 mRNA translation in a manner suggesting incomplete assembly of 48S preinitiation complexes (PICs) at upstream AUG codons in GCN4 mRNA. Rps5 mutations evoke accumulation of factors on native 40S subunits normally released on conversion of 48S PICs to 80S initiation complexes (ICs) and this abnormality and related phenotypes are mitigated by the SUI5 variant of eIF5. Remarkably, similar effects are observed by substitution of Lys45 in the Rps5-NTD, involved in contact with Rps16, and by eliminating the last two residues of the C-terminal tail (CTT) of Rps16, believed to contact initiator tRNA base-paired to AUG in the P site. We propose that Rps5-NTD-Rps16-NTD interaction modulates Rps16-CTT association with Met-tRNA_i^Met to promote a functional 48S PIC.     

Four N-linked glycosylation sites in human toll-like receptor 2 cooperate to direct efficient biosynthesis and secretion

Most higher organisms have a system of innate immune defense that is mediated by a group of evolutionarily related, germ line-encoded receptors, so-called Toll-like receptors. In mammals Toll-like receptors signal in response to pathogen-associated microbial structures. For example, Toll-like receptor 2 appears to mediate responses to bacterial peptidoglycan and acylated lipoproteins and Toll-like receptor 4 to bacterial lipopolysaccharide. However, the structural principles that underlie recognition of these structures are poorly understood. Toll-like receptors have leucine-rich repeats in their extracellular domains and are thus believed to adopt solenoid structures, similar to that found in platelet glycoprotein Ib. Additionally, all Toll-like receptors contain N-linked glycosylation consensus sites, and Toll-like receptor 4 requires glycosylation for function. Toll-like receptor glycosylation is also likely to influence receptor surface representation, trafficking, and pattern recognition. Using circular dichroism spectroscopy, we show here that purified human Toll-like receptor 2 and 4 proteins have secondary structure contents similar to glycoprotein Ib. We have also analyzed where consensus glycosylation sites are located in the extracellular domains of other human Toll-like receptors. We found that there are significant differences in the location and degree of conservation between sites in different Toll-like receptors. Using site-directed mutagenesis, we have found that in Toll-like receptor 2 extracellular domain all four predicted glycosylation sites are substituted, although one site is inefficiently core-glycosylated and its removal drastically affects secretion. The remaining Toll-like receptor 2 glycosylation sites also contribute to efficient protein secretion, albeit to a lesser degree.     

Site-specific DNA repair at the nucleosome level in a yeast minichromosome

The rate of excision repair of UV-induced pyrimidine dimers (PDs) was measured at specific sites in each strand of a yeast minichromosome containing an active gene (URA3), a replication origin (ARS1), and positioned nucleosomes. All six PD sites analyzed in the transcribed URA3 strand were repaired more rapidly (greater than 5-fold on average) than any of the nine PD sites analyzed in the nontranscribed strand. Efficient repair also occurred in both strands of a disrupted TRP1 gene (ten PD sites), containing four unstable nucleosomes, and in a nucleosome gap at the 5' end of URA3 (two PD sites). Conversely, slow repair occurred in both strands immediately downstream of the URA3 gene (12 of 14 PD sites). This region contains the ARS1 consensus sequence, a nucleosome gap, and two stable nucleosomes. Thus, modulation of DNA repair occurs in a simple yeast minichromosome and correlates with gene expression, nucleosome stability, and (possibly) control of replication.     

Expression,glycosylation and secretion of yeast acid phosphatase in hamster BHK cells

The gene PHO5 coding for one of the repressible acid phosphatases of the yeastSaccharomyces cerevisiae has been expressed at high efficiency in the baby hamster kidney (BHK) cell line. The expression vector was constructed from PHO5 driven by the human -actin promoter and was transfected into BHK cells by the calcium phosphate method. The recombinant APase (r-APase) which was secreted in active form from the cells was estimated by SDS/polyacrylamide gel electrophoresis to have molecular massM _r=62000, indicating substitution of the polypeptide moiety by 2–3 asparagine-linked glycans. Analysis by sequential lectin affinity chromatography of glycopeptides obtained from r-APase with Pronase showed that the glycans are predominantly of the 2.2.4 triantennary and tetraantennary complex-type. These data suggest that the extensive glycosylation of yeast APase, which contains eight polymannose substituents, is not essential for secretion and expression of enzymatic activity of the transfected gene product.Abbreviations APase acid phosphatase - PBS phosphate buffered saline - TBS Tris buffered saline - con A concanavalin A - TCA Tetracarpidium conophorum agglutinin     

Septin filament formation is essential in budding yeast

Septins are GTP-binding proteins that form ordered, rod-like multimeric complexes and polymerize into filaments, but how such supramolecular structure is related to septin function was unclear. In Saccharomyces cerevisiae, four septins form an apolar hetero-octamer (Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11) that associates end-to-end to form filaments. We show that septin filament assembly displays previously unanticipated plasticity. Cells lacking Cdc10 or Cdc11 are able to divide because the now-exposed subunits (Cdc3 or Cdc12, respectively) retain an ability to homodimerize via their so-called G interface, thereby allowing for filament assembly. In such cdc10Δ and cdc11Δ cells, the remaining septins, like wild-type complexes, localize to the cortex at the bud neck and compartmentalize nonseptin factors, consistent with a diffusion barrier composed of continuous filaments in intimate contact with the plasma membrane. Conversely, Cdc10 or Cdc11 mutants that cannot self-associate, but "cap" Cdc3 or Cdc12, respectively, prevent filament formation, block cortical localization, and kill cells.     

NgBR is essential for endothelial cell glycosylation and vascular development

NgBR is a transmembrane protein identified as a Nogo‐B‐interacting protein and recently has been shown to be a subunit required for cis‐prenyltransferase (cisPTase) activity. To investigate the integrated role of NgBR in vascular development, we have characterized endothelial‐specific NgBR knockout embryos. Here, we show that endothelial‐specific NgBR knockout results in embryonic lethality due to vascular development defects in yolk sac and embryo proper. Loss of NgBR in endothelial cells reduces proliferation and promotes apoptosis of the cells largely through defects in the glycosylation of key endothelial proteins including VEGFR2, VE‐cadherin, and CD31, and defective glycosylation can be rescued by treatment with the end product of cisPTase activity, dolichol phosphate. Moreover, NgBR functions in endothelial cells during embryogenesis are Nogo‐B independent. These data uniquely show the importance of NgBR and protein glycosylation during vascular development.     

The essential C family DnaE polymerase is error-prone and efficient at lesion bypass

DnaE-type DNA polymerases belong to the C family of DNA polymerases and are responsible for chromosomal replication in prokaryotes. Like most closely related Gram-positive cells, Streptococcus pyogenes has two DnaE homologs Pol C and DnaE; both are essential to cell viability. Pol C is an established replicative polymerase, and DnaE has been proposed to serve a replicative role. In this report, we characterize S. pyogenes DnaE polymerase and find that it is highly error-prone. DnaE can bypass coding and noncoding lesions with high efficiency. Error-prone extension is accomplished by either of two pathways, template-primer misalignment or direct primer extension. The bypass of abasic sites is accomplished mainly through "dNTP-stabilized" misalignment of template, thereby generating (-1) deletions in the newly synthesized strand. This mechanism may be similar to the dNTP-stabilized misalignment mechanism used by the Y family of DNA polymerases and is the first example of lesion bypass and error-prone synthesis catalyzed by a C family polymerase. Thus, DnaE may function in an error-prone capacity that may be essential in Gram-positive cells but not Gram-negative cells, suggesting a fundamental difference in DNA metabolism between these two classes of bacteria.     

Site-specific recombination promotes plasmid amplification in yeast

All stable, naturally occurring circular yeast DNA plasmids contain a pair of long, nontandem inverted repeats that undergo frequent reciprocal recombination. This yields two plasmid inversion isomers that exist in the cell in equal numbers. In the 2 mu circle plasmid of S. cerevisiae such inversion is catalyzed by a plasmid-encoded site-specific recombinase, FLP. We show that the site-specific recombination system of 2 mu circle enables the plasmid to increase its mean intracellular copy number in yeast cells growing under nonselective conditions. This apparently occurs by a FLP-induced transient shift in the mode of replication from theta to double rolling circle as initially proposed by Futcher. This capability may ensure stable maintenance of the plasmid by enabling it to correct downward deviations in copy number that result from imprecision of the plasmid-encoded partitioning system.     

The stability of yeast invertase is not significantly influenced by glycosylation

Yeast invertase exists in two different forms. The cytoplasmic enzyme is nonglycosylated, whereas the external invertase contains about 50% carbohydrate of the high mannose type. The protein moieties of both enzymes are identical. The two invertases have been used previously as a model system to investigate the influence of covalently linked carbohydrate chains on the stability of large glycoproteins, and controversial results were obtained. Here, we measured thermal and denaturant-induced unfolding by various probes, such as the loss of enzymatic activity, and by the changes in absorbance and fluorescence. The ranges of stability of the two invertases were found to be essentially identical, indicating that the presence of a high amount of carbohydrate does not significantly contribute to the stability of external invertase. Earlier findings that invertase is stabilized by glycosylation could not be confirmed. The stability of this glycoprotein is apparently determined by the specific interactions of the folded polypeptide chain. Unlike the glycosylated form, the carbohydrate-free invertase is prone to aggregation in the denatured state at high temperature and in a partially unfolded form in the presence of intermediate concentrations of guanidinium chloride.     

Fission yeast MO25 protein is localized at SPB and septum and is essential for cell morphogenesis

Cell morphogenesis is of fundamental significance in all eukaryotes for development, differentiation, and cell proliferation. In fission yeast, Drosophila Furry-like Mor2 plays an essential role in cell morphogenesis in concert with the NDR/Tricornered kinase Orb6. Mutations of these genes result in the loss of cell polarity. Here we show that the conserved proteins, MO25-like Pmo25, GC kinase Nak1, Mor2, and Orb6, constitute a morphogenesis network that is important for polarity control and cell separation. Intriguingly, Pmo25 was localized at the mitotic spindle pole bodies (SPBs) and then underwent translocation to the dividing medial region upon cytokinesis. Pmo25 formed a complex with Nak1 and was required for both the localization and kinase activity of Nak1. Pmo25 and Nak1 in turn were essential for Orb6 kinase activity. Further, the Pmo25 localization at the SPBs and the Nak1-Orb6 kinase activities during interphase were under the control of the Cdc7 and Sid1 kinases in the septation initiation network (SIN), suggesting a functional linkage between SIN and the network for cell morphogenesis/separation following cytokinesis.     

Site-specific glycosylation of human recombinant erythropoietin: analysis of glycopeptides or peptides at each glycosylation site by fast atom bombardment mass spectrometry

We have previously determined the carbohydrate structure of human recombinant erythropoietin [Sasaki, H., Bothner, B., Dell, A., & Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076]. The carbohydrate chains are distributed in three N-glycosylation sites and one O-glycosylation site. In order to examine the extent to which protein structure influences glycosylation, we have analyzed the saccharide structures at each glycosylation site (Asn24, Asn38, Asn83, and Ser126) of human recombinant erythropoietin. By high-performance liquid chromatography, we have succeeded in separation of glycopeptides containing different O-linked saccharides to the same peptide backbone. Fast atom bombardment mass spectrometry of the isolated glycopeptides combined with Edman degradation allowed us to elucidate the composition of glycopeptides and the amino acid attachment site. The analysis of glycopeptides and saccharides by fast atom bombardment mass spectrometry and high-performance liquid chromatography provided the following conclusions on N-glycans: (1) saccharides at Asn24 are heterogeneous and consist of biantennary, triantennary, and tetraantennary saccharides with or without N-acetyllactosaminyl repeats; (2) saccharides at Asn38 mainly consist of well-processed saccharides such as tetraantennary saccharides with or without N-acetyllactosaminyl repeats; (3) saccharides at Asn83, on the other hand, are homogeneous in the backbone structure and are composed mainly of tetraantennary without N-acetyllactosaminyl repeats. It was also noted that saccharides at Asn24 are much less sialylated than those at Asn38, although these two glycosylation sites are close to each other. These results clearly indicate that the protein structure and, possibly, the carbohydrate chain at the neighboring site greatly influence glycosylation of a given glycosylation site.     

The polyubiquitin gene is essential for meiosis in fission yeast

We isolated a novel sporulation-deficient mutant of Schizosaccharomyces pombe. The mutant did not have a mitotic growth defect but aborted meiosis at the first or the second division with condensed chromosomes that failed to separate, abnormal spindle(s), and disintegrated spindle pole bodies (SPBs). During the first division, the centromeres were pulled to near the spindle poles but condensed divalent chromosomes remained at the center. The failure to proceed to anaphase was also observed during a time-lapse recording of a SPB protein tagged with green fluorescent protein. The polyubiquitin gene ubi4(+), which encoded eight ubiquitins fused in tandem, complemented this mutant. The mutation, an A to G substitution, was identified within the ubi4(+) gene at the ATG initiation codon. Disruption of the ubi4(+) gene produced the same phenotypes. The ubi4(+) mRNA was strongly induced for meiosis. However, ubiquitin increases only slightly, suggesting that the role of the polyubiquitin gene is to supply ubiquitin that is consumed by unidentified mechanisms. Before the ubi4 mutant cells entered meiosis, ubiquitin was greatly decreased indicating that shortage of ubiquitin caused abortion of meiosis. This work provides insights for the role of polyubiquitin gene and importance of ubiquitination in SPB integrity at the meiotic divisions.     

The plug domain of yeast Sec61p is important for efficient protein translocation, but is not essential for cell viability

The Sec61/SecY translocon mediates translocation of proteins across the membrane and integration of membrane proteins into the lipid bilayer. The structure of the translocon revealed a plug domain blocking the pore on the lumenal side. It was proposed to be important for gating the protein conducting channel and for maintaining the permeability barrier in its unoccupied state. Here, we analyzed in yeast the effect of introducing destabilizing point mutations in the plug domain or of its partial or complete deletion. Unexpectedly, even when the entire plug domain was deleted, cells were viable without growth phenotype. They showed an effect on signal sequence orientation of diagnostic signal-anchor proteins, a minor defect in cotranslational and a significant deficiency in posttranslational translocation. Steady-state levels of the mutant protein were reduced, and when coexpressed with wild-type Sec61p, the mutant lacking the plug competed poorly for complex partners. The results suggest that the plug is unlikely to be important for sealing the translocation pore in yeast but that it plays a role in stabilizing Sec61p during translocon formation.     

Multiple cell culture factors can affect the glycosylation of Asn-184 in CHO-produced tissue-type plasminogen activator

Human tissue-type plasminogen activator (t-PA) contains a variably occupied glycosylation site at Asn-184 in naturally produced t-PA and in t-PA produced in recombinant Chinese hamster ovary (CHO) cells. The presence of an oligosaccharide at this site has previously been shown to reduce specific activity and fibrin binding. In this report, the site occupancy of t-PA is shown to increase gradually over the course of batch and fed-batch CHO cultures. Additional cell culture factors, including butyrate and temperature, are also shown to influence the degree of glycosylation. In each of these cases, conditions with decreased growth rate correlate with increased site occupancy. Investigations using quinidine and thymidine to manipulate the cell cycle distribution of cultures further support this correlation between site occupancy and growth state. Comparison of the cell cycle distribution across the range of cell culture factors investigated shows a consistent relationship between site occupancy and the fraction of cells in the G(0)/G(1) phase of the cell cycle. These results support a correlation between growth state and site occupancy, which fundamentally differs from site occupancy trends previously observed and illustrates the importance of the growth profile of CHO cultures in producing consistently glycosylated recombinant glycoproteins.     

The yeast LATS/Ndr kinase Cbk1 regulates growth via Golgi-dependent glycosylation and secretion

Saccharomyces cerevisiae Cbk1 is a LATS/Ndr protein kinase and a downstream component of the regulation of Ace2 and morphogenesis (RAM) signaling network. Cbk1 and the RAM network are required for cellular morphogenesis, cell separation, and maintenance of cell integrity. Here, we examine the phenotypes of conditional cbk1 mutants to determine the essential function of Cbk1. Cbk1 inhibition severely disrupts growth and protein secretion, and triggers the Swe1-dependent morphogenesis checkpoint. Cbk1 inhibition also delays the polarity establishment of the exocytosis regulators Rab-GTPase Sec4 and its exchange factor Sec2, but it does not interfere with actin polarity establishment. Cbk1 binds to and phosphorylates Sec2, suggesting that it regulates Sec4-dependent exocytosis. Intriguingly, Cbk1 inhibition causes a >30% decrease in post-Golgi vesicle accumulation in late secretion mutants, indicating that Cbk1 also functions upstream of Sec2-Sec4, perhaps at the level of the Golgi. In agreement, conditional cbk1 mutants mislocalize the cis-Golgi mannosyltransferase Och1, are hypersensitive to the aminoglycoside hygromycin B, and exhibit diminished invertase and Sim1 glycosylation. Significantly, the conditional lethality and hygromycin B sensitivity of cbk1 mutants are suppressed by moderate overexpression of several Golgi mannosyltransferases. These data suggest that an important function for Cbk1 and the RAM signaling network is to regulate growth and secretion via Golgi and Sec2/Sec4-dependent processes.     

Covalent flavinylation is essential for efficient redox catalysis in vanillyl-alcohol oxidase

By mutating the target residue of covalent flavinylation in vanillyl-alcohol oxidase, the functional role of the histidyl-FAD bond was studied. Three His(422) mutants (H422A, H422T, and H422C) were purified, which all contained tightly but noncovalently bound FAD. Steady state kinetics revealed that the mutants have retained enzyme activity, although the turnover rates have decreased by 1 order of magnitude. Stopped-flow analysis showed that the H422A mutant is still able to form a stable binary complex of reduced enzyme and a quinone methide product intermediate, a crucial step during vanillyl-alcohol oxidase-mediated catalysis. The only significant change in the catalytic cycle of the H422A mutant is a marked decrease in reduction rate. Redox potentials of both wild type and H422A vanillyl-alcohol oxidase have been determined. During reduction of H422A, a large portion of the neutral flavin semiquinone is observed. Using suitable reference dyes, the redox potentials for the two one-electron couples have been determined: -17 and -113 mV. Reduction of wild type enzyme did not result in any formation of flavin semiquinone and revealed a remarkably high redox potential of +55 mV. The marked decrease in redox potential caused by the missing covalent histidyl-FAD bond is reflected in the reduced rate of substrate-mediated flavin reduction limiting the turnover rate. Elucidation of the crystal structure of the H422A mutant established that deletion of the histidyl-FAD bond did not result in any significant structural changes. These results clearly indicate that covalent interaction of the isoalloxazine ring with the protein moiety can markedly increase the redox potential of the flavin cofactor, thereby facilitating redox catalysis. Thus, formation of a histidyl-FAD bond in specific flavoenzymes might have evolved as a way to contribute to the enhancement of their oxidative power.