Mechanistic insights into N-glycosylation microheterogeneity of recombinant proteins produced in cell culture

Please click Additional Files below to see the full abstract

The yeast integral membrane protein Apq12 potentially links membrane dynamics to assembly of nuclear pore complexes

Although the structure and function of components of the nuclear pore complex (NPC) have been the focus of many studies, relatively little is known about NPC biogenesis. In this study, we report that Apq12 is required for efficient NPC biogenesis in Saccharomyces cerevisiae. Apq12 is an integral membrane protein of the nuclear envelope (NE) and endoplasmic reticulum. Cells lacking Apq12 are cold sensitive for growth, and a subset of their nucleoporins (Nups), those that are primarily components of the cytoplasmic fibrils of the NPC, mislocalize to the cytoplasm. APQ12 deletion also causes defects in NE morphology. In the absence of Apq12, most NPCs appear to be associated with the inner but not the outer nuclear membrane. Low levels of benzyl alcohol, which increases membrane fluidity, prevented Nup mislocalization and restored the proper localization of Nups that had accumulated in cytoplasmic foci upon a shift to lower temperature. Thus, Apq12p connects nuclear pore biogenesis to the dynamics of the NE

Complementation of Essential Yeast GPI Mannosyltransferase Mutations Suggests a Novel Specificity for Certain <i>Trypanosoma</i> and <i>Plasmodium</i> PigB Proteins

<div><p>The glycosylphosphatidylinositol (GPI) anchor is an essential glycolipid that tethers certain eukaryotic proteins to the cell surface. The core structure of the GPI anchor is remarkably well conserved across evolution and consists of NH₂-CH₂-CH₂-PO₄-6Manα1,2Manα1,6Manα1,4-GlcNα1,6-<i>myo</i>-inositol-PO₄-lipid. The glycan portion of this structure may be modified with various side-branching sugars or other compounds that are heterogeneous and differ from organism to organism. One such modification is an α(1,2)-linked fourth mannose (Man-IV) that is side-branched to the third mannose (Man-III) of the trimannosyl core. In fungi and mammals, addition of Man-III and Man-IV occurs by two distinct Family 22 α(1,2)-mannosyltransferases, Gpi10/PigB and Smp3/PigZ, respectively. However, in the five protozoan parasite genomes we examined, no genes encoding Smp3/PigZ proteins were observed, despite reports of tetramannosyl-GPI structures (Man₄-GPIs) being produced by some parasites. In this study, we tested the hypothesis that the Gpi10/PigB proteins produced by protozoan parasites have the ability to add both Man-III and Man-IV to GPI precursors. We used yeast genetics to test the <i>in vivo</i> specificity of Gpi10/PigB proteins from several <i>Plasmodium</i> and <i>Trypanosoma</i> species by examining their ability to restore viability to <i>Saccharomyces cerevisiae</i> strains harboring lethal defects in Man-III (<i>gpi10</i>Δ) or Man-IV (<i>smp3</i>Δ) addition to GPI precursor lipids. We demonstrate that genes encoding PigB enzymes from <i>T. cruzi</i>, <i>T. congolense</i> and <i>P. falciparum</i> are each capable of separately complementing essential <i>gpi10</i>Δ and <i>smp3</i>Δ mutations, while <i>PIGB</i> genes from <i>T. vivax</i> and <i>T. brucei</i> only complement <i>gpi10</i>Δ. Additionally, we show the ability of <i>T. cruzi PIGB</i> to robustly complement a <i>gpi10</i>Δ/<i>smp3</i>Δ double mutant. Our data suggest that certain <i>Plasmodium</i> and <i>Trypanosoma</i> PigB mannosyltransferases can transfer more than one mannose to GPI precursors <i>in vivo</i>, and suggest a novel biosynthetic mechanism by which Man₄-GPIs may be synthesized in these organisms.</p></div

Modifications of the GPI anchor core structure observed in various organisms¹.

<p>Chart describing known side chain modifications of the GPI core in various species. R1-R6 and Lipid positions on the GPI core are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087673#pone-0087673-g001" target="_blank">Figure 1</a>. DAG: diacylglycerol; AAG: alkylacylglycerol; DMG: dimyristylglycerol; Man: mannose; EthNP: ethanolamine phosphate; GalNAc: N-acetylgalactosamine; AEP: aminoethylphosphonate; Cer: ceramide.</p>1<p>See references 7, 10, 16, 17, 20</p

Schematic of the core GPI anchor glycan and its side chain modifications.

<p>GPI anchors consist of a highly conserved carbohydrate sequence that is linked to the inositol head group (hexagon) of phosphatidylinositol (hexagon + LIPID). The core GPI glycan contains glucosamine (blue/white square) and three mannoses (Man I-III, green circles). A protein is linked to the third mannose via an ethanolamine phosphate. R indicates positions where modification of the core GPI structure can occur, and possible R modifications in various species are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087673#pone-0087673-t001" target="_blank">Table 1</a>. The red R₁ indicates the position of fourth mannose addition.</p

Complementation of yeast <i>gpi10</i> and <i>smp3</i> null mutants by protozoan <i>PIGB</i> genes.

<p>A. Schematic of yeast plasmid shuffling experiments. Haploid <i>gpi10</i>::Kan^R containing pGAL<i>-ScGPI10</i> (top row) or <i>smp3</i>::Kan^R containing pGAL<i>-ScSMP3</i> (bottom row) strains were transformed with protozoan pPGK<i>-PIGB</i> expression plasmids. Yeasts carrying both plasmids were then induced to lose one plasmid via growth on rich medium, and then strains containing only the pPGK plasmids were selected for by growth on 5FOA. B. Serial drop cultures of yeast on SGal and SD + 5FOA media. Note that all strains grow on SGal medium as they contain both the pGAL rescuing plasmid and the pPGK expression plasmid. Following selection on 5FOA only the strains containing <i>PIGB</i> genes that can complement the indicated null mutation are able to grow.</p

<i>T. cruzi PIGB</i> can complement a yeast strain deficient in both <i>gpi10</i> and <i>smp3</i>.

<p>A. Schematic of a tetrad showing the desired non-parental ditype pattern of kanamycin marker segregation generated from the mating of a haploid <i>gpi10</i>::Kan^R strain containing pGAL-<i>TcrPIGB</i> with a haploid <i>smp3</i>::Kan^R strain containing pGAL-<i>TcrPIGB</i>. B. PCR was used to test each haploid meiotic segregant from a non-parental ditype tetrad for the presence of <i>gpi10</i>Δ and <i>smp3</i>Δ alleles (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087673#pone-0087673-g003" target="_blank">Figure 3B</a> for PCR schematic). Haploids 1 and 2 are each double mutants that contain both null alleles. C. Serial drop cultures of both single <i>gpi10</i>Δ and <i>smp3</i>Δ mutant strains as well as the double mutant <i>gpi10</i>Δ<i>/smp3</i>Δ strain. Note that the strains grow on the galactose-containing medium that induces pGAL-<i>TcrPIGB</i> expression but not on the glucose medium, which represses pGAL-<i>TcrPIGB</i> expression.</p

The long and short conserved motifs do not confer broad specificity to <i>Tcr</i>PigB.

<p>A. Alignment of the N-terminal long and C-terminal short motifs from yeast and protozoa. B. Schematics of the generated chimera constructs. The N-terminal long (chimera 1), C-terminal short (chimera 2) or both conserved motifs (chimera 3) of <i>TcrPIGB</i> (red) were replaced by those from <i>TbPIGB</i> (green). Numbers correspond to the amino acid positions of each domain within its native protein sequence. C. Serial drop cultures of control and chimeric constructs in both <i>gpi10</i>Δ and <i>smp3</i>Δ strains before (SGal) and after (SD + 5FOA) plasmid shuffling. Note that all three of the chimeric PigB proteins are able to complement the <i>gpi10</i>Δ and <i>smp3</i>Δ mutant strains.</p

PCR confirmation of yeast strains.

<p>A–C. Schematics of PCR reactions used to confirm strains. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087673#pone.0087673.s003" target="_blank">Table S3</a> for sequences of the indicated primers. A. To confirm the presence (on SGal) or absence (on 5FOA) of the pGAL rescuing plasmid, the <i>URA3</i> locus within this plasmid was amplified. B. To confirm that the endogenous <i>gpi10</i> or <i>smp3</i> locus was indeed null in all strains, forward primers residing in the 5′UTR of the genes were coupled with a reverse primer that sits within the kanamycin cassette that was inserted into the gene locus upon null strain generation. C. To confirm the presence of the pPGK plasmid, PCR was performed using a primer within the pPGK vector, coupled with a primer in the 3′end of the coding region of the <i>PIGB</i> gene of interest. D–E. Results of PCR experiments to confirm all yeast strains shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087673#pone-0087673-g002" target="_blank">Figure 2</a>. DNA was isolated from all strains and the PCRs described in A–C were performed. D. <i>gpi10</i>Δ strains before (SGal) and after (5FOA) plasmid shuffling. E. <i>smp3</i>Δ strains before (SGal) and after (5FOA) plasmid shuffling. Reactions A–C correspond to the schematics A–C at left. Amplicon sizes are: Reaction A: 500 bp; Reaction B: 534 bp for <i>gpi10</i>, 616 bp for <i>smp3</i>; Reaction C: 902 bp for pPGK-415, 891 bp for pPGK-<i>ScGPI10</i>, 922 bp for pPGK-<i>ScSMP3</i>, 800 bp for pPGK-<i>TbPIGB</i>, 820 bp for pPGK-<i>TcrPIGB</i>, 879 bp for pPGK-<i>TvPIGB</i>, 932 bp for pPGK-<i>TcoPIGB</i> and 908 bp for pPGK-<i>PfPIGB.</i></p