Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain

Interactions with cell surface receptors enhance the therapeutic properties of many important antibodies, including the low-affinity Fc γ Receptors (FcγRs). These interactions require proper processing of the immunoglobulin G Fc N-glycan, and eliminating the N-glycan abolishes binding, restricting antibody production to mammalian expression platforms. Yeasts, for example, generate extensively mannosylated N-glycans that are unsuitable for therapeutics. However, Fc with a specifically truncated N-glycan still engages receptors with considerable affinity. Here we describe the creation and applications of a novel Saccharomyces cerevisiae strain that specifically modifies the IgG1 Fc domain with an N-glycan consisting of a single N-acetylglucosamine residue. This strain displayed glycoengineered Fc on its surface for screening yeast surface display libraries and also served as an alternative platform to produce glycoengineered Rituximab. An IgG-specific endoglycosidase (EndoS2) truncates the IgG1 Fc N-glycan. EndoS2 was targeted to the yeast ER using the signal peptide from the yeast protein disulfide isomerase (PDI) and a yeast ER retention signal (HDEL). Furthermore, >99% of the yeast expressed Rituximab displayed the truncated glycoform as determined by SDS-PAGE and ESI-MS analyses. Lastly, the yeast expressed Rituximab engaged the FcγRIIIa with the expected affinity (KD = 2.0 ± 0.5 μM) and bound CD20 on Raji B cells.

Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function

Shuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells and tissues derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis.

Interaction of Pestiviral E1 and E2 Sequences in Dimer Formation and Intracellular Retention

Pestiviruses contain three envelope proteins: Erns, E1, and E2. Expression of HA-tagged E1 or mutants thereof showed that E1 forms homodimers and -trimers. C123 and, to a lesser extent, C171, affected the oligomerization of E1 with a double mutant C123S/C171S preventing oligomerization completely. E1 also establishes disulfide linked heterodimers with E2, which are crucial for the recovery of infectious viruses. Co-expression analyses with the HA-tagged E1 wt/E1 mutants and E2 wt/E2 mutants demonstrated that C123 in E1 and C295 in E2 are the critical sites for E1/E2 heterodimer formation. Introduction of mutations preventing E1/E2 heterodimer formation into the full-length infectious clone of BVDV CP7 prevented the recovery of infectious viruses, proving that C123 in E1 and C295 in E2 play an essential role in the BVDV life cycle, and further support the conclusion that heterodimer formation is the crucial step. Interestingly, we found that the retention signal of E1 is mandatory for intracellular localization of the heterodimer, so that absence of the E1 retention signal directs the heterodimer to the cell surface even though the E2 retention signal is still present. The covalent linkage between E1 and E2 plays an essential role for this process.

A region within the third extracellular loop of rat Aquaporin 6 precludes trafficking to plasma membrane in a heterologous cell line

The inability to over-express Aquaporin 6 (AQP6) in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail using crystallographic approaches. Using an Aquaporin 3-tGFP Reporter (AGR) system we have identified a region within loop C of AQP6 that is responsible for severely hampering plasma membrane expression. Serine substitution corroborated that amino acids present within AQP6194–213 of AQP6 loop C contribute to intracellular endoplasmic reticulum (ER) retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous expression systems.

Production of Gloeophyllum trabeum Endoglucanase Cel12A in Nicotiana benthamiana for Cellulose Degradation

Lignocellulosic biomass from plants has been used as a biofuel source and the potent acidic endoglucanase GtCel12A has been isolated from Gloeophyllum trabeum, a filamentous fungus. In this study, we established a plant-based platform for the production of active GtCel12A fused to family 3 cellulose-binding module (CBM3). We used the signal sequence of binding immunoglobulin protein (BiP) and the endoplasmic reticulum (ER) retention signal for the accumulation of the produced GtCel12A in the ER. To achieve enhanced enzyme expression, we incorporated the M-domain of the human receptor-type tyrosine-protein phosphatase C into the construct. In addition, to enable the removal of N-terminal domains that are not necessary after protein expression, we further incorporated the cleavage site of Brachypodium distachyon small ubiquitin-like modifier. The GtCel12A-CBM3 fusion protein produced in the leaves of Nicotiana benthamiana exhibited not only high solubility but also efficient endoglucanase activity on the carboxymethyl cellulose substrate as determined by 3,5-dinitrosalicylic acid assay. The endoglucanase activity of GtCel12A-CBM3 was maintained even when immobilized on microcrystalline cellulose beads. Taken together, these results indicate that GtCel12A endoglucanase produced in plants might be used to provide monomeric sugars from lignocellulosic biomass for bioethanol production.

Characterization of membrane topology and retention signal of pestiviral glycoprotein E1

Pestiviruses are members of the family Flaviviridae, a group of enveloped viruses that bud at intracellular membranes. Pestivirus particles contain three glycosylated envelope proteins, Erns, E1 and E2. Among them, E1 is the least characterized concerning both biochemical features and function. E1 from bovine viral diarrhea virus (BVDV) strain CP7 was analyzed with regard to its intracellular localization and membrane topology. Here, it is shown that even in the absence of other viral proteins, E1 is not secreted or expressed at the cell surface, but localizes predominantly in the ER. Using engineered chimeric TM domains with sequences from E1 and vesicular stomatitis virus G protein, the E1 ER-retention signal could be narrowed down to six fully conserved polar residues in the middle part of the transmembrane domain of E1. Retention was observed even when several of these polar residues were exchanged for alanine. Mutations with a strong impact on E1 retention prevented recovery of infectious viruses when tested in the viral context. Analysis of the membrane topology of E1 before and after the signal peptide cleavage via a selective permeabilization and an in vivo labelling approach revealed that mature E1 is a typical type I transmembrane protein with a single span transmembrane anchor at its C-terminus whereas it adopts a hairpin-like structure with the C-terminus located in the ER lumen when the pre-cleavage situation is mimicked by blocking the cleavage site between E1 and E2. Importance The shortage of specific antibodies against E1 making detection and further analysis of E1 difficult, resulted in a lack of knowledge on E1 compared to Erns and E2 with regard to biosynthesis, structure and function. It is known that pestiviruses bud intracellularly. Here, we show that E1 contains its own ER retention signal: six fully conserved polar residues in the middle part of the TM domain are shown to be the determinants for ER retention of E1. Moreover, those six polar residues could serve as a functional group that intensely affect the generation of infectious viral particles. In addition, the membrane topology of E1 has been determined. In this context, we also identified dynamic changes in membrane topology of E1 with the carboxy-terminus located on the luminal side of the ER in the pre-cleavage state and relocation of this sequence upon signal peptidase cleavage. Our work provides the first systematic analysis of the pestiviral E1 protein with regard to its biochemical and functional characteristics.

A Region Within the Third Extracellular Loop of Rat AQP6 Precludes Its Trafficking to Plasma Membrane in a Heterologous Cell Line.

The inability to over-express AQP6 in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail. Using the AGR reporter system we have identified a region within loop C of AQP6 that is responsible for severely hampering its plasma membrane localization. Serine substitution corroborated that amino acids present within AQP6194-213 of AQP6 loop C contribute to intracellular retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous cells.

Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function

Shuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis.

Unconventional ER to Endosomal Trafficking by a Retroviral Protein

ABSTRACTMouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the ER membrane into a functional N-terminal signal peptide (SP) and the C-terminus (Rem-CT). Rem-CT lacks a membrane-spanning domain and a known ER retention signal, yet was not detectably secreted into cell supernatants. Inhibition of intracellular trafficking by the drug Brefeldin A (BFA), which interferes with the ER to Golgi secretory pathway, resulted in dramatically reduced intracellular Rem-CT levels. A Rem mutant lacking glycosylation sites was cleaved into SP and Rem-CT, but was insensitive to BFA, suggesting that unglycosylated Rem-CT does not exit the ER or reach a degradative compartment. BFA reduction of Rem-CT levels was not rescued by proteasome or lysosomal inhibitors. Rem-CT has simple glycans, which are necessary for Rem-CT stability and trafficking, but indicate that Rem-CT does not traffic through the Golgi. Analysis of wild-type Rem-CT and its glycosylation mutant by confocal microscopy revealed that both were primarily localized to the ER lumen. A small fraction of wild-type Rem-CT, but not the unglycosylated mutant, were co-localized with Rab5+ endosomes. Expression of a dominant-negative (DN) form of ADP ribosylation factor 1 (Arf1) (T31N) mimicked the effects of BFA by reducing Rem-CT levels, suggesting that Arf1 prevents Rem-CT localization to a degradative compartment. A DN form of the AAA ATPase, p97/VCP, rescued Rem-CT in the presence of BFA or DN Arf1. Thus, Rem-CT uses an unconventional trafficking scheme, perhaps to thwart innate immunity to MMTV infection.IMPORTANCEMouse mammary tumor virus is a complex retrovirus that encodes a regulatory/accessory protein, Rem. Rem is a precursor protein that is processed at the endoplasmic reticulum (ER) membrane by signal peptidase. The N-terminal SP eludes ER-associated degradation to traffic to the nucleus and serve a human immunodeficiency virus Rev-like function. In contrast, the function of the C-terminal glycosylated cleavage product (Rem-CT) is unknown. Since localization is critical for protein function, we used multiple methods to localize Rem-CT. Surprisingly, Rem-CT, which lacks a transmembrane domain or an ER retention signal, was detected primarily within the ER and required glycosylation for trafficking to endosomes. Blocking of retrograde trafficking through Arf1 reduced Rem-CT levels, but was not restored by lysosomal or proteasomal inhibitors. The unique trafficking of Rem-CT suggests a novel intracellular trafficking pathway, potentially impacting host anti-viral immunity.

Therapeutic Targeting of CCR1 to Prevent Dissemination of Multiple Myeloma Plasma Cells

Introduction:Multiple myeloma (MM) disease progression is dependent on the ability of the MM plasma cells (PC) to leave the bone marrow (BM), re-enter the peripheral blood (PB) and disseminate to other BM sites. Previous studies show that expression of CXCL12 by BM stromal cells is crucial for MM PC retention within the BM. However, the mechanisms which overcome this retention signal enabling MM PC egress and dissemination via the PB are poorly understood. Previous studies in haematopoietic progenitor cells have demonstrated that CCL3 overcomes the CXCL12 retention signal to drive mobilisation to the PB (Lord et al. Blood 1995). Here, we examined the role of the CCL3 chemokine receptor CCR1 in driving MM PC dissemination. Methods and results: Initially, we assessed the expression of CCR1 protein on CD138+CD38++CD45loCD19- PC from 28 MM, 8 MGUS and 2 SMM patients by flow cytometry. Results show CCR1 expression is significantly increased in newly diagnosed MM compared with premalignant MGUS and SMM patients (p=0.03; CCR1 MFI mean±SEM, MGUS: 53.0±33.6; SMM: 37.6±8.9 MM: 250.9±71.6). Furthermore, CCR1 expression on PB MM PC positively correlated with PB MM PC numbers (p=0.03; n=11 patients). To identify mechanistically how CCR1 may promote dissemination, the effect of CCL3 on the response to CXCL12 in human myeloma cell lines (HMCL) was assessed in vitro. The migration of RPMI-8226 and OPM2 cells was induced by CCL3 or CXCL12 chemoattractant in a transwell assay. Notably, pre-treatment of RPMI-8226 or OPM2 with CCL3 abrogated migration towards CXCL12 and blocked F-actin remodelling in response to CXCL12 in vitro. These findings suggest that CCL3 can desensitise cells to exogenous CXCL12, providing a potential mechanism facilitating loss of the CXCL12 retention signal. To confirm whether CCR1 is required for driving MM PC dissemination, homozygous CCR1 knockout (KO) cells were generated using a lentiviral CRISPR/Cas9 system in OPM2 cells. CCR1-KO OPM2 cells were confirmed to have no detectable CCR1 expression by flow cytometry and could no longer migrate towards CCL3 in vitro. Empty vector (EV) or CCR1-KO OPM2 MM PC were injected into the tibia of immune-compromised NOD-scidgamma (NSG) mice. After 4 weeks, primary tumour within the injected tibia and disseminated tumour in the PB and the contralateral tibia and femur was assessed by flow cytometry. We found that mice bearing CCR1-KO cells have a 45.5% decrease in primary tumour growth (p=0.008; % GFP+ of total mononuclear cells, EV: 77.2±17.2; CCR1-KO: 42.1±24.4), a 97.8% reduction in PB MM PC (p<0.0001; EV: 1.39±0.7; CCR1-KO: 0.03±0.046) anda 99.9% reduction in BM tumour dissemination (p<0.0001; EV: 49.5±17; CCR1-KO: 0.019±0.013), compared with controls. In a supportive study, CCR1 was expressed in the murine MM cell line 5TGM1 using lentiviral transduction. 5TGM1-CCR1 cells were confirmed to express CCR1 by qPCR and were able to migrate towards CCL3 in vitro. 5TGM1-CCR1 or EV cells were injected into the tibiae of C57BL/KaLwRij mice and allowed to initiate systemic MM disease for 3.5 weeks. Importantly, while 55% of control mice exhibited disseminated tumours, this increased to 92% with CCR1 expression (p<0.0001; n=12/group). These data suggest that CCR1 expression on MM PC may play an important role in MM PC dissemination. To determine whether therapeutic inhibition of CCR1 prevents dissemination, the effect of a small molecule CCR1 inhibitor, CCR1i, was assessed in vivo. OPM2 EV or RPMI-8226 cells were injected into the tibia of NSG mice and, after 3 days, mice were treated with CCR1i (15mg/kg) or vehicle twice daily by oral gavage for 25 days. OPM2-inoculated CCR1i-treated mice had 66.1% lower PB MM PC (p<0.0001; % GFP+ of total mononuclear cells, vehicle: 23.9±7.2; CCR1i: 8.1±3.8) and a 22.1% reduction in BM dissemination (p=0.0002; vehicle: 78.1±4.8;CCR1i: 60.8±7.1) compared with controls. Similarly, CCR1i treatment reduced BM dissemination by 59.6% in RPMI-8226 bearing mice (p<0.0001; % GFP+ of total mononuclear cells, vehicle: 0.86±0.15; CCR1i: 0.26±0.05). This suggests that CCR1 inhibition can slow tumour dissemination in vivo. Conclusion:This study identified CCR1 as a novel driver of MM PC dissemination in vivo, at least in part by overcoming the CXCL12 retention signal. Importantly, this study demonstrated for the first time that targeting CCR1 can be a viable therapeutic strategy to limit dissemination and potentially slow disease progression. Disclosures Croucher: Trovagene: Employment.