Potential Glycosylation Sites Research Articles

The diversity of glycosylation of cellobiohydrolase I from Trichoderma reesei determined with mass spectrometry

Cellulases are glycosylated enzymes that have wide applications in fields like biofuels. It has been widely accepted that glycosylation of cellulases impact their performance. Trichoderma reesei is the most important cellulase-producer and cellobiohydrolase I (CBHI) is the most important cellulase from T. reesei. Therefore, the glycosylation of T. reesei CBHI has been a focus of research. However, investigations have been focused on N-glycosylation of three of the four potential glycosylation sites, as well as O-glycosylation on the linker region, while a full picture of glycosylation of T. reesei CBHI is still needed. In this work, with extensive mass spectrometric investigations on CBHI from two T. reesei strains grown under three conditions, several new discoveries were made: 1) N45 and N64 are N-glycosylated with high mannose type glycans; 2) the catalytic domain of CBHI is extensively O-glycosylated with hexoses and N-acetylhexosamines; 3) experimental evidence on the mannosylation of carbohydrate binding domain (other than the linker adjacent region) was found. With structural analysis, we found several glycosylation sites (such as T383, S8, and S46) are located at the openings of the substrate-binding tunnel, and potentially involve in the binding of cellulose. These investigations provide a full and comprehensive picture on the glycosylation of CBHI from T. reesei, which benefits the engineering of CBHI by raising potential sites for modification.

Structural glycoprotein LmAbd-9 is required for the formation of the endocuticle during locust molting

Insect cuticle is a composite made of chitin filaments embedded in a proteinaceous matrix, consisting mainly of structural cuticular proteins. In the present study, an endocuticle structural glycoprotein gene, LmAbd-9, was characterized based on the Locusta migratoria transcriptome. LmAbd-9 encodes a glycoprotein with a chitin binding domain 4, belonging to RR-1 subclass of the CPR family, which has two potential O-linked glycosylation sites (S115 and T137) at which glycosylation modification may occur. LmAbd-9 was highly expressed in the integument and showed periodic expression during molting. The expression levels of LmAbd-9 were significantly down-regulated after injection with 20-hydroxyecdysone (20E) for 6, 12, and 24 h, whereas it was upregulated after double-stranded RNA-mediated RNA interference of the 20E receptor gene LmEcR and LmFTZ-F1beta at day 2 of fifth instar nymphs for 48 h. After injection of dsLmAbd-9 on day 2 of fifth instar nymphs, the insects could normally molt to adults and showed no macroscopic phenotype; however, the cuticle of the adults was thinner, and there were significantly fewer endocuticular lamellae than in the control. Thus, LmAbd-9 that negatively regulated by the 20E signaling pathway was involved in the formation of the endocuticle in L. migratoria.

Complement 1q-binding protein from Nile tilapia (Oreochromis niloticus): Molecular characterization, expression pattern upon bacterial infection and its binding properties

Complement 1q-binding protein (gC1qR), a multi-functional cellular protein, is able to bind the globular head region of C1q molecule (gC1q), playing an important role in regulation of immune response and host defense against bacterial infection. In this study, the full length of a gC1qR ortholog (OngC1qR) was identified and characterized from Nile tilapia (Oreochromis niloticus). The cDNA of OngC1qR ORFs consisted of 870 bp of nucleotide sequence encoding a 289 amino acid residue polypeptide. The deduced amino acid sequence is highly homologous to teleost, containing a MAM33p domain, two potential glycosylation sites, and a putative cell adhesion site EGD (Glu-Gly-Asp). Spatial mRNA expression analysis revealed that the OngC1qR was expressed ubiquitously in all examined tissues, with a high expression in the liver. The OngC1qR expression was significantly up-regulated in liver, spleen and head kidney following in vivo challenges with Streptococcus agalactiae. A high up-regulation was also detected in head kidney leukocytes in vitro stimulation with Streptococcus agalactiae. In addition, immunofluorescence detection illustrated that the OngC1qR was located at the cell surface of leukocytes. Recombinant OngC1qR protein was able to not only bind LPS, LTA, S. agalactiae and Aeromonas hydrophila, but also possess binding capability with the ligand OnC1qs. Taken together, the results indicated that OngC1qR might be involved in host defense against bacterial infection in Nile tilapia.

First characterizations by capillary electrophoresis of human Chorionic Gonadotropin at the intact level

In the present work, the first characterizations by Capillary Electrophoresis of the human Chorionic Gonadotropin (hCG) hormone at the intact level were carried out. hCG is a hetero-dimeric glycoprotein, specific to the human pregnancy, consisting of an α and a β subunit, so-called hCGα and hCGβ, respectively. hCG has 8 potential glycosylation sites leading to a high number of isoforms (including glycoforms and other post-translational modifications) that we are interesting to characterize. First, Capillary Gel Electrophoresis (CGE) was used to separate the isoforms of two hCG-based drugs: Ovitrelle® (recombinant r-hCG) and Pregnyl (hCG isolated from the urine of pregnant women, u-hCG). As expected, CGE led to a better resolution than SDS-PAGE and confirmed the large heterogeneity of hCG. Different CGE profiles were obtained for the two hCG-based drugs, varying in number of peaks, migration times, and peak intensities, thus demonstrating that the drugs contain isoforms, different in nature and proportion. This result was confirmed by Capillary IsoElectrophoretic Focusing (CIEF). The pI ranges of the hCG isoforms were found between 3.4 and 4.7, and 4.5 and 5.2 for r-hCG and u-hCG, respectively. This information was further used to develop the separation of the hCG isoforms by Capillary Zone Electrophoresis (CZE). The pH, the nature, and the concentration of the background electrolyte as well as the nature and the content of its organic modifier were optimized. The use of a coated capillary to avoid protein adsorption was also evaluated. The final CZE-UV method allowed distinguishing at least 6 peaks, corresponding to different hCG isoforms.To significantly improve the level of information obtained, the CZE instrument was then coupled by means of an electrospray ionization source to a triple quadrupole (TQ) mass spectrometer. Two detection strategies were used, one focusing on the lower m/z values (100–1000) in order to identify some sugar residues as diagnostic ions to confirm the presence of glycan chains, and the second focusing on the higher m/z values (1000–2000), corresponding to the multiple charged intact protein isoforms. For both approaches, the fragmentor and capillary voltage values were optimized. The composition and the flow-rate of the sheath liquid were then optimized for the strategy focusing on the higher m/z values in order to both increase the charge state of the ionized intact isoforms and the signal-to-noise ratio. The final method was used to compare the two hCG-based drugs, demonstrating the potential of the developed CZE-MS method for isoforms fingerprinting.

PROBLEMS OF ISOLATION, IDENTIFICATION AND ANTIGENIC CHARACTERIZATION OF RECENT HUMAN A(H3N2) INFLUENZA VIRUSES.

Human A (H3N2) influenza viruses are distinguished by a high rate of evolution and regularly cause epidemics around the world. Their ability to adapt and to escape from the host's immune response and to change their receptor specificity is very high. Over the past 20 years, these viruses have lost the ability to agglutinate red blood cells of chickens and turkeys and have practically ceased to propagate in chicken embryos - the main source of influenza vaccines. Isolation of viruses in the MDCK cell culture led to the selection of strains that lose one of the potential glycosylation sites. Many of the A (H3N2) strains have acquired mutations in neuraminidase, which distort the results of antigenic analysis in the hemagglutination inhibition test - the cornerstone method for the analysis of the match between viral isolates circulating in human population to strains selected for the influenza vaccines. In this regard, the characteristics of the antigenic properties of influenza A (H3N2) viruses by traditional methods become poorly informative, and the selection of vaccine strains of this subtype is erroneous, which is reflected in the discrepancy between vaccine and circulating A (H3N2) viruses in recent years (2013-2014, 2014 -2015, 2015-2016). The search, development and implementation of new algorithms for the isolation and antigen analysis of influenza A (H3N2) viruses are extremely urgent.

Characterization of UDP-Activated Purinergic Receptor P2Y6 Involved in Japanese Flounder Paralichthys olivaceus Innate Immunity

Uridine 5’-diphosphate (UDP)-activated purinergic receptor P2Y6 is a member of a G-protein-coupled purinergic receptor family that plays an important role in mammalian innate immunity. However, the role of the P2Y6 receptor (P2Y6R) in fish immunity has not been investigated. In this report, we characterized a P2Y6R gene from Japanese flounder (Paralichthys olivaceus) and examined its role in fish innate immunity. Sequence analysis reveals that the Japanese flounder P2Y6R protein is conserved and possesses four potential glycosylation sites. Quantitative real-time RT-PCR analysis shows that P2Y6R is broadly distributed in all examined Japanese flounder tissues with dominant expression in the liver. In addition, P2Y6R gene expression was up-regulated in head kidney macrophages (HKMs) upon lipopolysaccharides (LPS) and poly(I:C) stimulations but down-regulated by LPS challenge in peripheral blood leukocytes (PBLs). Furthermore, pharmacological inhibition of the endogenous P2Y6 receptor activity by the potently selective P2Y6R antagonist, MRS 2578, greatly up-regulated pro-inflammatory cytokine interleukin (IL)-1β, IL-6 and TNF-α gene expression in PBL cells treated with UDP. Moreover, LPS- and poly(I:C)-induced gene expression of IL-1β and TNF-α in Japanese flounder PBL cells was attenuated significantly by inhibition of P2Y6R activity with antagonist MRS 2578. Collectively, we, for the first time, showed the involvement of functional purinergic P2Y6R in fish innate immunity.

Characterization of neutralizing epitopes in antigenic site B of recently circulating influenza A(H3N2) viruses.

Influenza A(H3N2) viruses are associated with outbreaks worldwide and can cause disease with severe complications. The impact can be reduced by vaccination, which induces neutralizing antibodies that mainly target the haemagglutinin glycoprotein (HA). In this study we generated neutralizing mouse monoclonal antibodies (mAbs) against A/Victoria/361/2011 and identified their epitopes by generating and sequencing escape viruses. The epitopes are located in antigenic site B, which is near the receptor-binding site and is immunodominant in humans. Amino acid (aa) substitutions at positions 156, 158, 159, 189, 190 and 193 in antigenic site B led to reduced ability of mAbs to block receptor-binding. The majority of A(H3N2) viruses that have been circulating since 2014 are antigenically distinct from previous A(H3N2) viruses. The neutralization-sensitive epitopes in antigenic site B of currently circulating viruses were examined with these mAbs. We found that clade 3C.2a viruses, possessing an additional potential glycosylation site at HA1 position N158, were poorly recognized by some of the mAbs, but other residues, notably at position 159, also affected antibody binding. Through a mass spectrometric (MS) analysis of HA, the glycosylated sites of HA1 were established and we determined that residue 158 of HA1 was glycosylated and so modified a neutralization-sensitive epitope. Understanding and monitoring individual epitopes is likely to improve vaccine strain selection.

A spliced form of CD44 expresses the unique glycan that is recognized by the prostate cancer specific antibody F77.

Prostate cancer is the most common cancer occurring in men in the United States. The monoclonal antibody F77 that was originally developed in our laboratory recognizes mainly glycolipids as well as O-linked glycosylation on proteins in prostate cancer cells. We have identified a spliced form of glycoprotein CD44 as one critical protein expressing the F77 antigen. The F77-specific glycosylation occurs on multiple potential glycosylation sites on the CD44 protein encoded by the fourteenth exon. CD44 is a tumor stem cell marker and is known to induce tumor stemness and metastasis. Knockdown of CD44 or FUT1 genes dramatically reduced F77-induced apoptosis in prostate cancer cell lines. We developed an ELISA using both a CD44 antibody and F77 to identify the special form of glycosylated CD44 from prostate cancer cells as well as from serum samples of prostate cancer patients. These results reveal a CD44-dependent mechanism for F77 to induce tumor cell apoptosis, and a new strategy for the detection of glycosylated CD44 proteins secreted by prostate cancer cells.

Site-Directed Mutagenesis of the Fibronectin Domains in Insulin Receptor-Related Receptor.

The orphan insulin receptor-related receptor (IRR), in contrast to its close homologs, the insulin receptor (IR) and insulin-like growth factor receptor (IGF-IR) can be activated by mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by its extracellular region, involves multiple domains, and shows positive cooperativity with two synergistic sites. By the analyses of point mutants and chimeras of IRR with IR in, we now address the role of the fibronectin type III (FnIII) repeats in the IRR pH-sensing. The first activation site includes the intrinsically disordered subdomain ID (646–716) within the FnIII-2 domain at the C-terminus of IRR alpha subunit together with closely located residues L135, G188, R244, H318, and K319 of L1 and C domains of the second subunit. The second site involves residue T582 of FnIII-1 domain at the top of IRR lambda-shape pyramid together with M406, V407, and D408 from L2 domain within the second subunit. A possible importance of the IRR carbohydrate moiety for its activation was also assessed. IRR is normally less glycosylated than IR and IGF-IR. Swapping both FnIII-2 and FnIII-3 IRR domains with those of IR shifted beta-subunit mass from 68 kDa for IRR to about 100 kDa due to increased glycosylation and abolished the IRR pH response. However, mutations of four asparagine residues, potential glycosylation sites in chimera IRR with swapped FnIII-2/3 domains of IR, decreased the chimera glycosylation and resulted in a partial restoration of IRR pH-sensing activity, suggesting that the extensive glycosylation of FnIII-2/3 provides steric hindrance for the alkali-induced rearrangement of the IRR ectodomain.

Site-specific glycosylation profile of influenza A (H1N1) hemagglutinin through tandem mass spectrometry

ABSTRACTThe study of influenza virus evolution in humans has revealed a significant role of glycosylation profile alterations in the viral glycoproteins – hemagglutinin (HA) and neuraminidase (NA), in the emergence of both seasonal and pandemic strains. Viral antigenic drift can modify the number and location of glycosylation sites, altering a wide range of biological activities and the antigenic properties of the strain. In view of the key role of glycans in determining antigenicity, elucidating the glycosylation profiles of influenza strains is a requirement towards the development of improved vaccines. Sequence-based analysis of viral RNA has provided great insight into the role of glycosite modifications in altering virulence and pathogenicity. Nonetheless, this sequence-based approach can only predict potential glycosylation sites. Due to experimental challenges, experimental confirmation of the occupation of predicted glycosylation sites has only been carried out for a few strains. Herein, we utilized HCD/CID-MS/MS tandem mass spectrometry to characterize the site-specific profile of HA of an egg-grown H1N1 reference strain (A/New Caledonia/20/1999). We confirmed experimentally the occupancy of glycosylation sites identified by primary sequence analysis and determined the heterogeneity of glycan structures. Four glycosylation sequons on the stalk region (N28, N40, N304 and N498) and four on the globular head (N71, N104, N142 and N177) of the protein are occupied. Our results revealed a broad glycan microheterogeneity, i.e., a great diversity of glycan compositions present on each glycosite. The present methodology can be applied to characterize other viruses, particularly different influenza strains, to better understand the impact of glycosylation on biological activities and aid the improvement of influenza vaccines.

Cell attenuated porcine epidemic diarrhea virus strain Zhejiang08 provides effective immune protection attributed to dendritic cell stimulation.

Since 2010, the porcine epidemic diarrhea coronavirus (PEDV) has caused significant damage to the global pork industry. However, classical PEDV vaccine strains only provide limited protection against emerging strains. In this study, we successfully isolated and attenuated the PEDV epidemic strain Zhejiang08, which was characterized by good cell adaptation and high-titer production 48 h post infection in Vero E6 cells. The attenuated virus induced a high level of virus-specific neutralizing antibodies until 120 days after immunization in piglets and provided complete protection when challenged with an emerging virus strain on day 14 post immunization. Moreover, the capability to activate dendritic cells (DCs) of this isolate was identified. Higher expression levels of IL-12 and IFN-γ were recorded in DCs after treatment with Zhejiang08 for 24 h. Furthermore, genome sequencing and phylogenetic analysis revealed high homology between the main antigen epitopes of Zhejiang08 and PEDV pandemic isolates following 2011. Combining the glycosylation site prediction results and their distribution within the spatial structure of the S protein, led to the conclusion that the observed more effective host immune response of Zhejiang08 compared to CV777 was possibly associated with a lack of the potential glycosylation site in the 296 amino acids of the S protein. In summary, we illustrated that the attenuated virus represents a promising vaccine candidate.

Establishment of CMab-43, a Sensitive and Specific Anti-CD133 Monoclonal Antibody, for Immunohistochemistry.

CD133, also known as prominin-1, was first described as a cell surface marker on early progenitor and hematopoietic stem cells. It is a five-domain transmembrane protein composed of an N-terminal extracellular tail, two small cytoplasmic loops, two large extracellular loops containing seven potential glycosylation sites, and a short C-terminal intracellular tail. CD133 has been used as a marker to identify cancer stem cells derived from primary solid tumors and as a prognostic marker of gliomas. Herein, we developed novel anti-CD133 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We expressed the full length of CD133 in LN229 glioblastoma cells, immunized mice with LN229/CD133 cells, and performed the first screening using flow cytometry. After limiting dilution, we established 100 anti-CD133 mAbs, reacting with LN229/CD133 cells but not with LN229 cells. Subsequently, we performed the second and third screening with Western blot and immunohistochemical analyses, respectively. Among 100 mAbs, 11 strongly reacted with CD133 in Western blot analysis. One of 11 clones, CMab-43 (IgG2a, kappa), showed a sensitive and specific reaction against colon cancer cells, warranting the use of CMab-43 in detecting CD133 in pathological analyses of CD133-expressing cancers.

TcPHYLL, a cacao phylloplanin expressed in young tissues and glandular trichomes

Phylloplanins are potential antimicrobial compounds that can be found on the surface of some plants. These compounds were initially identified in Nicotiana tabacum as proteins secreted by short glandular trichomes (STG), which play an important role in controlling infection caused by Peronospora tabacina, especially by acting on the phylloplane, the first site of plant-pathogen interaction. A protein similar to phylloplanins, containing an Ole e 1 domain, was identified in the Theobroma cacao genome database. In this study, we analyzed the Open Read Frame (ORF) and the promoter of T. Cacao phylloplanin (Tcphyll) using bioinformatics tools, the expression pattern of its transcript by RT-qPCR and the promoter activity driving the β-glucuronidase (GUS) expression in transgenic tobacco. The results indicate that Tcphyll encodes a protein of 158 amino acid residues, with signal peptide and potential glycosylation and phosphorylation sites, which are grouped with phylloplanins instead of allergenic pollen grain proteins. Lower levels of Tcphyll transcripts were found in flowers, and higher levels were found in shoot apical meristem, 2–3 cm segment with leaf primordia and, rich in glandular trichomes. Accumulation of transcripts in meristems increased on the first day after inoculation with Moniliophthora perniciosa, and it was reduced after the second day. Promoter analysis identified 40 cis-acting regulatory elements; the GUS expression, under the control of 731 base pairs of the Tcphyll promoter, was observed in tall and short glandular trichomes. The results indicate that TcPHYLL is the first characterized phylloplanin from a perennial tree.

Comparison of proteomic profiles of the venoms of two of the ‘Big Four’ snakes of India, the Indian cobra (Naja naja) and the common krait (Bungarus caeruleus), and analyses of their toxins

Snake venoms are mixtures of biologically-active proteins and peptides, and several studies have described the characteristics of some of these toxins. However, complete proteomic profiling of the venoms of many snake species has not yet been done. The Indian cobra (Naja naja) and common krait (Bungarus caeruleus) are elapid snake species that are among the ‘Big Four’ responsible for the majority of human snake envenomation cases in India. As understanding the composition and complexity of venoms is necessary for successful treatment of envenomation in humans, we utilized three different proteomic profiling approaches to characterize these venoms: i) one-dimensional SDS-PAGE coupled with in-gel tryptic digestion and electrospray tandem mass spectrometry (ESI-LC-MS/MS) of individual protein bands; ii) in-solution tryptic digestion of crude venoms coupled with ESI-LC-MS/MS; and iii) separation by gel-filtration chromatography coupled with tryptic digestion and ESI-LC-MS/MS of separated fractions. From the generated data, 81 and 46 different proteins were identified from N. naja and B. caeruleus venoms, respectively, belonging to fifteen different protein families. Venoms from both species were found to contain a variety of phospholipases A2 and three-finger toxins, whereas relatively higher numbers of snake venom metalloproteinases were found in N. naja compared to B. caeruleus venom. The analyses also identified less represented venom proteins including L-amino acid oxidases, cysteine-rich secretory proteins, 5’-nucleotidases and venom nerve growth factors. Further, Kunitz-type serine protease inhibitors, cobra venom factors, phosphodiesterases, vespryns and aminopeptidases were identified in the N. naja venom, while acetylcholinesterases and hyaluronidases were found in the B. caeruleus venom. We further analyzed protein coverage (Lys/Arg rich and poor regions as well as potential glycosylation sites) using in-house software. These studies expand our understanding of the proteomes of the venoms of these two medically-important species.

Expression and functional characterization of vitronectin gene from Japanese flounder (Paralichthys olivaceus)

Vitronectin (Vtn) is a multifunctional protein that plays significant roles in cell adhesion, migration, spreading and survival, and in the regulation of membrane attack complex formation and the terminal pathway of complement activation in innate immune response. However, the expression and immune significance of Vtn in fish remains largely unknown. In order to understand the involvement of Vtn in fish innate immune response, here we cloned and characterized a full-length Vtn ortholog cDNA, termed PoVtn, from Japanese flounder Paralichthys olivaceus. The deduced PoVtn protein is comprised of 438 amino acids with a 19-amino-acid signal peptide sequence (1Met-19Ala) at the N-terminus. Protein domain analysis revealed that PoVtn possesses a conserved N-terminal somatomedin B domain followed by a conserved RGD motif and four haemopexin-like domains. Sequence analysis revealed that PoVtn has two potential glycosylation sites and shares 44–74% sequence identity with other teleost Vtn proteins. PoVtn mRNA was ubiquitously distributed in all examined normal tissues and showed the highest expression in Japanese flounder hepatopancreas tissue. PoVtn expression was induced by LPS and poly(I:C) challenges in the Japanese flounder head kidney macrophages (HKMs) and peripheral blood leucocytes (PBLs) and shows a pathogen-associated molecular pattern- and cell type-dependent manner. The expression of PoVtn was also modulated by bacterial challenge with Edwardsiella tarda in Japanese flounder immune-related tissues including head kidney, gill and spleen. Furthermore, overexpression of PoVtn in Japanese flounder FG-9307 cells significantly attenuated the LPS- and poly(I:C)-induced proinflammatory cytokines IL-1beta and TNF-alpha gene expression. Taken our findings together, we for the first time characterized Vtn gene expression in response to inflammatory stimuli in fish. Our results suggested a potential role of PoVtn in regulating fish innate immunity.

Structural modeling of human organic cation transporters

Human organic cation transporters (hOCTs) belong to solute carriers (SLC) 22 family of membrane proteins that play a central role in transportation of chemotherapeutic drugs for several clinical and pathological conditions, including cancer and diabetes. These transporters mediate drug transport; however, the precise mechanism of drug-binding and transport by them is not fully uncovered yet, partly due to unavailability of any crystal structure record. In this work, we performed a multi-phasic approach to compute the 3D structural models of seven human organic cation transporters (hOCTs) starting from primary protein sequence. Our structure modeling approach included 1) I-TASSER based comparative sequence alignment, threading and ab-initio protein modeling; 2) models comparison with PSIPRED secondary structure prediction; 3) loop modeling for incongruent secondary structure in Chimera 1.10.1; 4) high resolution structure simulation, refinement, energy minimization using ModRefiner, and 5) validation of the structure models using PROCHECK at SAVEs. From structural point, the computed 3D structures of hOCTs consist of a typical major facilitator superfamily (MFS) fold of twelve α-transmembrane helix domains arranged in a manner rendering hOCTs a barrel shaped structure with a large cleft that opens in cytoplasm. The modeled 3D structure of all hOCTs closely resemble to human SLC2A3 (GLUT3) transporter (PDB ID: 5c65) and displayed an outward-open confirmation and putative cyclic C1 protein symmetry. In addition, hOCTs has a large (>100 amino acids) unique extracellular loop between TMH1 and TMH2 having potential glycosylation sites (Asn-Xaa-Ser/Thr) and cysteine residues, both features indicative of putative role in drug binding and uptake. There is an intracellular three/four-helix loop between TMH6 and TMH7 containing putative phosphorylation sites for precise regulation of hOCTs function as drug transporters. There are nine loops of 4 to 11 amino acids length that protrude from membrane, both intracellularly and extracellularly, and connect adjacent TMHs. The 2D structure prediction showed Nin-Cin topology of all hOCTs. In the unavailability of the crystal structures of hOCTs, the 3D structural models computed in-silico and presented herein can be used for studying the mechanism of drug binding and transport by hOCTs.

Emergence of a novel subclade of influenza A(H3N2) virus in London, December 2016 to January 2017.

We report the molecular investigations of a large influenza A(H3N2) outbreak, in a season characterised by sharp increase in influenza admissions since December 2016. Analysis of haemagglutinin (HA) sequences demonstrated co-circulation of multiple clades (3C.3a, 3C.2a and 3C.2a1). Most variants fell into a novel subclade (proposed as 3C.2a2); they possessed four unique amino acid substitutions in the HA protein and loss of a potential glycosylation site. These changes potentially modify the H3N2 strain antigenicity.

N-Glycoprofiling Analysis for Carbohydrate Composition and Site-Occupancy Determination in a Poly-Glycosylated Protein: Human Thyrotropin of Different Origins

Human thyrotropin (hTSH) is a glycoprotein with three potential glycosylation sites: two in the α-subunit and one in the β-subunit. These sites are not always occupied and occupancy is frequently neglected in glycoprotein characterization, even though it is related to folding, trafficking, initiation of inflammation and host defense, as well as congenital disorders of glycosylation (CDG). For the first time N-glycoprofiling analysis was applied to the site-occupancy determination of two native pituitary hTSH, in comparison with three recombinant preparations of hTSH, a widely used biopharmaceutical. A single methodology provided the: (i) average N-glycan mass; (ii) mass fraction of each monosaccharide and of sulfate; and (iii) percent carbohydrate. The results indicate that the occupancy (65%–87%) and carbohydrate mass (12%–19%) can be up to 34%–57% higher in recombinant hormones. The average glycan mass is 24% lower in pituitary hTSH and contains ~3-fold fewer moles of galactose (p < 0.005) and sialic acid (p < 0.01). One of the two native preparations, which had the smallest glycan mass together with the lowest occupancy and GalNAc, sulfate, Gal and sialic acid contents, also presented the lowest in vivo bioactivity and circulatory half-life. The methodology described, comparing a recombinant biopharmaceutical to its native equivalent, can be applied to any physiologically or clinical relevant glycoprotein.

Breast milk and in utero transmission of HIV-1 select for envelope variants with unique molecular signatures

BackgroundMother-to-child transmission of human immunodeficiency virus-type 1 (HIV-1) poses a serious health threat in developing countries, and adequate interventions are as yet unrealized. HIV-1 infection is frequently initiated by a single founder viral variant, but the factors that influence particular variant selection are poorly understood.ResultsOur analysis of 647 full-length HIV-1 subtype C and G viral envelope sequences from 22 mother–infant pairs reveals unique genotypic and phenotypic signatures that depend upon transmission route. Relative to maternal strains, intrauterine HIV transmission selects infant variants that have shorter, less-glycosylated V1 loops that are more resistant to soluble CD4 (sCD4) neutralization. Transmission through breastfeeding selects for variants with fewer potential glycosylation sites in gp41, are more sensitive to the broadly neutralizing antibodies PG9 and PG16, and that bind sCD4 with reduced cooperativity. Furthermore, experiments with Affinofile cells indicate that infant viruses, regardless of transmission route, require increased levels of surface CD4 receptor for productive infection.ConclusionsThese data provide the first evidence for transmission route-specific selection of HIV-1 variants, potentially informing therapeutic strategies and vaccine designs that can be tailored to specific modes of vertical HIV transmission.

Global N-Glycan Site Occupancy of HIV-1 gp120 by Metabolic Engineering and High-Resolution Intact Mass Spectrometry.

A vital step in HIV vaccine development strategies has been the observation that some infected individuals generate broadly neutralizing antibodies that target the glycans on the surface of HIV-1 gp120. These antibodies target glycan epitopes on viral envelope spikes, and yet the positions and degree of occupancy of glycosylation sites is diverse. Therefore, there is a need to understand glycosylation occupancy on recombinant immunogens. The sheer number of potential glycosylation sites and degree of chemical heterogeneity impedes assessing the global sequon occupancy of gp120 glycoforms. Here, we trap the glycan processing of recombinant gp120 to generate homogeneous glycoforms, facilitating occupancy assessment by intact mass spectrometry. We show that gp120 monomers of the BG505 strain contain either fully occupied sequons or missing the equivalent of one and sometimes two glycans across the molecule. This biosynthetic engineering approach enables the analysis of therapeutically important glycoproteins otherwise recalcitrant to analysis by native mass spectrometry.