Differentiating the structure of isobaric glycopeptides represents a major challenge for mass spectrometry-based characterisation techniques. Here we show that the regiochemistry of the most common N-acetylneuraminic acid linkages of N-glycans can be identified in a site-specific manner from individual glycopeptides using ion mobility-mass spectrometry analysis of diagnostic fragment ions.

(2015) Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation, mAbs, 7:5, 863-870, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Keywords: core fucosylation, sialylation, Nicotiana benthamiana, bisected glycans, glycan modelling, IgG, cetuximab Abbreviations: 3-FucT, Zea maize core a1,3-fucosyltransferase;3-FucT GnTIV, human a1,3-mannosyl-b1,4-N-acetyL-glucosaminyltransferase fused to the CTS region of the Arabidopsis thaliana core a1,3-fucosyltransferase (FUT11); 6-FucT, Mus musculus core a1,6-fucosyltransferase; CH2, constant domain of an IgG heavy chain; CTS, cytoplasmic tail, transmembrane domain and stem region; CxMab cetuximab (Erbitux Ò ); Fab, fragment, antigen-binding; Fc, Fragment crystallizable region of immunoglobulin G; GlcNAc, N-acetylglucosamine; IgG1, Immunoglobulin G subclass 1; LC-ESI-MS, Liquid chromatography-electrospray ionisationmass spectrometry; mAb, monoclonal antibody; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; ST GalT, b1,4-galactosyltransferase fused to the CTS region of the rat a2,6-sialyltransferase; 6-SiaT, a2,6-sialyltransferase;ST GnT-III, b1,4-mannosyl-b1,4-N-acetylglucosaminyltransferase fused to the CTS region of the rat a2,6-sialyltransferase; DXT/FT, Nicotiana benthamiana glycosylation mutants lacking plant specific core b1,2-xylose and a1,3-fucose residuesWe investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in the Fab domain in addition to the conserved Fc glycosylation, as a reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms of CxMab differing in core fucosylation (a1,3-and a1,6-linkage) were generated in a plant-based expression platform. These GnGn, GnGnF 3 , and GnGnF 6 CxMab variants were subjected in vivo to further processing toward sialylation and GlcNAc diversification (bisected and branching structures). Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans toward envisaged structures. By contrast, Fc glycan processing largely depend on the presence of core fucose. A particularly strong support of glycan processing in the presence of plant-specific core a1,3-fucose was observed. Consistently, molecular modeling suggests changes in the interactions of the Fc carbohydrate chain depending on the presence of core fucose, possibly changing the accessibility. Here, we provide data that reveal molecular mechanisms of glycan processing of IgG antibodies, which may have implications for the generation of glycan-engineered therapeutic antibodies with improved efficacies.

The importance of protein glycosylation in the biomedical field requires methods that not only quantitate structures by their monosaccharide composition, but also resolve and identify the many isomers expressed by mammalian cells. The art of unambiguous identification of isomeric structures in complex mixtures, however, did not yet catch up with the fast pace of advance of high-throughput glycomics. Here, we present a strategy for deducing structures with the help of a deci-minute accurate retention time library for porous graphitic carbon chromatography with mass spectrometric detection. We implemented the concept for the fundamental N-glycan type consisting of five hexoses, four Nacetylhexosamines and one fucose residue. Nearly all of the 40 biosynthetized isomers occupied unique elution positions. This result demonstrates the unique isomer selectivity of porous graphitic carbon. With the help of a rather tightly spaced grid of isotope-labeled internal N-glycan, standard retention times were transposed to a standard chromatogram. Application of this approach to animal and human brain N-glycans immediately identified the majority of structures as being of the bisected type. Most notably, it exposed hybrid-type glycans with galactosylated and even Lewis X containing bisected N-acetylglucosamine, which have not yet been discovered in a natural source. Thus, the time grid approach implemented herein facilitated discovery of the still missing pieces of the N-glycome in our most noble organ and suggests itselfin conjunction with collision induced dissociationas a starting point for the overdue development of isomer-specific deep structural glycomics.

An ideal method for the analysis of N-glycans would both identify the isomeric structure and deliver a true picture of the relative, if not absolute, amounts of the various structures in one sample. Porous graphitic carbon chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) detection has emerged as a method with a particularly high potential of resolving isomeric oligosaccharides, but little attention has so far been paid to quantitation of the results obtained. In this work, we isolated a range of structures from Man5 to complex type N-glycans with zero to four sialic acids and blended them into an equimolar “glyco tune mix”. When subjected to liquid chromatography–ESI-MS in positive and negative modes, the glyco tune mix clearly demonstrated the futility of quantitation of N-glycans of different overall composition, different number of sialic acids, and strongly differing size without compensation for their very different molar responses. Relative quantitation of human plasma N-glycans was performed with correction factors deduced from this external glyco tune mix. Addition of just one isotope-coded internal standard with enzymatically added 13C-galactose led to absolute quantification in the same experiment. Graphical AbstractDiscrepancy between desirable (grey bars) and real (green bars) relative ion abundance of equimolar amounts of glycans in positive mode ESI-MS. Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-017-0235-8) contains supplementary material, which is available to authorized users.

Microalgae of the genus Chlorella vulgaris are candidates for the production of lipids for biofuel production. Besides that, Chlorella vulgaris is marketed as protein and vitamin rich food additive. Its potential as a novel expression system for recombinant proteins inspired us to study its asparagine-linked oligosaccharides (N-glycans) by mass spectrometry, chromatography and gas chromatography. Oligomannosidic N-glycans with up to nine mannoses were the structures found in culture collection strains as well as several commercial products. These glycans co-eluted with plant N-glycans in the highly shape selective porous graphitic carbon chromatography. Thus, Chlorella vulgaris generates oligomannosidic N-glycans of the structural type known from land plants and animals. In fact, Man5 (Man5GlcNAc2) served as substrate for GlcNAc-transferase I and a trace of an endogenous structure with terminal GlcNAc was seen. The unusual more linear Man5 structure recently found on glycoproteins of Chlamydomonas reinhardtii occurred - if at all - in traces only. Notably, a majority of the oligomannosidic glycans was multiply O-methylated with 3-O-methyl and 3,6-di-O-methyl mannoses at the non-reducing termini. This modification has so far been neither found on plant nor vertebrate N-glycans. It’s possible immunogenicity raises concerns as to the use of C. vulgaris for production of pharmaceutical glycoproteins.

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.

Poxviruses are among the largest double-stranded DNA viruses with members such as Variola virus, Monkeypox virus and the famous vaccination strain Vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core, found in all infectious poxvirus forms, has remained sparse. While major core proteins have been annotated via indirect experimental evidence, their structures have remained elusive and they could not be assigned to the individual architectural features of the core. Hence, which proteins constitute which layers of the core, such as the so-called palisade layer and the inner core wall has remained enigmatic. Here, we have performed a multi-modal cryo-electron microscopy (cryo-EM) approach to elucidate the structural determinants of the VACV core. In combination with molecular modeling using AlphaFold, we unambiguously identify trimers formed by the cleavage product of A10 as the key component of the palisade layer. This allows us to place previously-obtained descriptions of protein interactions within the core wall into perspective and to provide a substantially revised model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely identical among Poxviridae, implying that our structural observations should be generalizable over most, if not all members of this important virus family.

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 was ∼100-fold more potent at promoting RSV mature CA assembly than observed for HIV-1 and removal of IP6 in vivo reduced infectivity by 100-fold. By cryo-electron tomography and subtomogram averaging, mature virus-like particles (VLPs) showed an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 had opposing effects on CA in vitro assembly, inducing formation of T=1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles revealed that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.