They must be exposed to the RhD antigen to develop the antibodies. Once this has happened, a second transfusion can result in a severe hemagglutination reaction. The child produces red blood cells with the RhD antigen, which gains access to the mother's circulation when the placenta ruptures during childbirth. Responding to the foreign RhD antigens, the mother produces anti-RhD antibodies that are able to cross the placenta.
Hemolytic disease causes liver and spleen enlargement resulting in decreased function, fluid accumulation, congestive heart failure and possibly death. This disease can be prevented by injecting the RhD - mother with Anti-RhD antibodies RhoGam within 72 hours after delivery of the first child.
In today's lab we will determine the ABO and Rh blood type of two different samples using a simulated human blood typing kit. Rachel Watson, M. AG Cell: rwatson uwyo. ABO Blood Grouping ABO blood grouping is based on differences in the type of glycoprotein protein with carbohydrates attached present on the surface of red blood cells.
The N protein is subsequently associated with the positive sense genomic RNA to become a nucleoprotein complex nucleocapsid , which together with S, M, and E proteins as well as other viral proteins, is further assembled and followed by budding into the lumen of the ER-Golgi intermediate compartment ERGIC to form mature virions. Finally, the mature virions are released from the host cell, waiting for a new life cycle to start.
The S1 and S2 subunits remain associated through noncovalent interactions in a metastable prefusion state Furin-like cleavage is essential for the S-protein mediated cell-cell fusion and viral infectivity, and is required for efficient SARS-CoV-2 infection of human lung cells 24 and airway epithelial cells Besides, a fraction of mature SARS-CoV-2 S proteins travel through the secretory pathway to the plasma membrane, where they can mediate fusion of infected with uninfected cells to form multinucleated giant cells syncytia 24 , Presumably, this deletion may enhance the cell surface expression of the SARS-CoV-2 S glycoprotein 32 , thereby facilitating the S protein incorporation into pseudovirions and replication-competent virions.
Mature S glycoprotein on the viral surface is a heavily glycosylated trimer, each protomer of which is composed of amino acids residues Figure 2A. Arrows denote protease cleavage sites. One protomer is shown in ribbon representation colored corresponding to the schematic in A , a second protomer in light gray surface representation, and the third protomer in dark gray surface representation. The glycans were omitted for clarity. Like other class I viral fusion proteins, the SARS-CoV-2 S glycoprotein is also a conformational machine that mediates viral entry by rearranging from a metastable unliganded state, through a pre-hairpin intermediate state, to a stable postfusion state 38 , The structural information provides a blueprint for structure-based design of vaccine immunogens and entry inhibitors of SARS-CoV The S1 subunits rest above the S2 trimer, stabilizing the later in the prefusion conformation Figure 2B This will be beneficial to the dissociation of the S1 subunit and facilitate conformational rearrangements that the S2 trimer undergoes to mediate viral entry.
Notably, it is well established that trimeric prefusion HIV-1 Env primarily resides in a closed configuration that is conformationally masked to evade antibody-mediated neutralization 43 , 44 and can spontaneously sample a transient, functional configuration It can thus be speculated that native CoV S glycoproteins on mature and infectious virions share a similar conformational masking feature 46 , concealing the receptor-binding surface for those utilizing CTDs as RBDs Figure 2C , which is further discussed below.
Structurally, RBD consists of two subdomains: a core and an external subdomain 51 , As the interaction between the RBD and ACE2 is extensive, small molecules probably cannot be used as entry inhibitors to effectively block the virus entry by targeting the interaction interface. However, peptides would be able to engage most of the residues belonging to RBM The formation of a trimer-of-hairpins structure also known as six-helix bundle comprising HR1 and HR2 in the postfusion conformation is a unifying feature of class I viral fusion proteins Three HR1 helices form a parallel central coiled-coil with three HR2 helices packing in an oblique, antiparallel manner against deep hydrophobic grooves on the surface of the central coiled-coil Notably, when a full-length S protein construct bearing the native furin-like cleavage site was transiently expressed by ExpiF cells, the purified S proteins contained the dissociated S2 trimer in the postfusion conformation The SARS-CoV-2 S trimer in the pre-hairpin intermediate state is very unstable and is just transiently present in vivo after triggering by ACE2 engagement, stymieing structural characterization of the S protein in this state However, although this fusion-intermediate phase is very short, it is enough for inhibitory peptides to associate with the pre-hairpin intermediate and block the six-helix bundle formation Furthermore, it has already been shown that the HR1 regions in various human CoVs are highly conserved 61 , and therefore could serve as an attractive target for the design and development of potent and broad-spectrum inhibitors of pan-CoVs, including SARS-CoV The SARS-CoV-2 S sequence encodes up to 22 N-linked glycan sequons per protomer, which likely plays an important role in protein folding 19 and host immune evasion as a glycan shield Of the 22 potential N-linked glycosylation sites on the S protein, 14 were identified to be predominantly occupied by processed, complex-type glycans The remaining eight sites were found to be dominated by oligomannose-type glycans, which are divergent from those founded on host glycoproteins When the site-specific N-linked glycans are mapped onto the prefusion structure of the SARS-CoV-2 S ectodomain 63 , the resulting model exhibited substantially higher levels of glycan-free surface than that revealed by structures of fully glycosylated, trimeric HIV-1 Env ectodomains 65 , These observations suggest that carbohydrate moieties could be immunogenic and highlight the need for immunogens to display the glycans important for the recognition of neutralizing antibodies 73 ; in support of this, specific N-linked glycans on Hemagglutinin has been shown to be essential for the elicitation of broadly neutralizing antibodies against Influenza Accordingly, there has been mounting interest in exploring the potential of immunogenic glycan moieties as vaccine candidates against multiple viruses, including SARS-CoV-2 75 , Following the second cleavage, the fusion peptide at the N terminus of the S2 trimer is inserted into the host membrane 8 , forming the pre-hairpin intermediate state Since the pre-hairpin intermediate state is extremely unstable, the S2 fusion protein is refolded quickly and irreversibly into the stable postfusion state 39 , These large conformational rearrangements pull the viral and host cell membrane into close proximity, leading ultimately to the membrane fusion 8 , Most of these vaccine strategies are based on the full-length S glycoprotein, the major viral surface antigen When a vaccine strategy requires that the SARS-CoV-2 S protein be recombinantly expressed in the human body, the ERRS should be omitted to enhance the cell surface expression level of the resulting protein.
Theoretically, the native HIV-1 Env trimer present on the surface of intact virions is thought to be a most ideal immunogen 60 , as most of the neutralizing antibodies thus far described could recognize and bind to the prefusion form of trimeric HIV-1 Env, although it is with great difficulty that such neutralizing antibodies against this glycan-covered, sequence-variable native form are induced For SARS-CoV-2, different lines of research have shown that convalescent sera from SARS-CoV and SARS-CoV-2 patients showed no or limited cross-neutralization activity against these two viruses by pseudotyped and authentic viral infection assays, despite significant cross-reactivity in binding to the S glycoproteins of both viruses 9 , 79 — Similar results were also observed in infected or immunized animals 48 , 79 , Therefore, SARS-CoV-2 evades immune surveillance also through conformational masking, which is well-documented for HIV-1 43 , 44 ; while at the same time, the S protein could transiently sample the functional state to engage ACE2, consistent with the notion that the fusion glycoprotein of highly pathogenic viruses have evolved to perform its functions while evading host neutralizing antibody responses.
Another concern for vaccine candidates based on the full-length S glycoprotein of SARS-CoV-2 is raised by the observation that the S1 subunit could spontaneously dissociate from the S glycoprotein probably as a trimer that still assumes the RBD closed conformation, leaving only the postfusion S2 trimer The resulting S1 and S2 subunits might expose immunodominant, nonneutralizing epitopes that are utilized by SARS-CoV-2 to serve as decoys to distract the host immune system, inducing a large proportion of ineffective antibody responses, as documented for HIV-1 60 and respiratory syncytial virus RSV Therefore, although the S proteins of both SARS-CoV and SARS-CoV-2 are thought to be promising vaccine immunogens for generating protective immunity, optimizing antigen design is critical to ensure an optimal immune response through exposing more neutralizing epitopes and displaying fewer potentially weakly or non-neutralizing epitopes This minimal form of RBDs of both viruses could serve as a vaccine candidate However, a conserved cysteine residue is located immediately upstream of the minimal RBD fragments of both viruses and always forms a disulfide bond in nearly all published structures containing this residue , ; this is also the case for Middle East respiratory syndrome coronavirus MERS-CoV , and HCoV-HKU1 37 , consistent with the observation that all RBDs of these viruses share a conserved structural core.
The disulfide bond contributes to stabilization of the RBD structure and likely modulates the protein immunogenicity. This notion is consistent with the observation that mice immunized with a longer form of the SARS-CoV RBD residues produced a higher titer of neutralizing antibodies compared with mice immunized with the minimal RBD region residues Besides the RBD, which has been shown to a major target for human neutralizing antibody responses , the NTD was recently identified to be a new vulnerable site of the SARS-CoV-2 S protein for antibody neutralizing and therefore could also serve as a recombinant protein-based vaccine — As expected, NTD-specific neutralizing antibodies could target the S protein in both closed and open conformations In addition, the apparent accessibility of the fusion peptide and HR1 region in published structures of the SARS-CoV-2 S ectodomain trimer as well as their high sequence conservation among CoVs suggests that they would be good immunogen candidates for epitope-focused vaccine design aimed at raising broadly CoV neutralizing antibodies The epitope-focused vaccine design has proven to be successful in generating neutralizing antibodies against RSV fusion glycoprotein However, neutralizing antibodies targeted against these two regions still need to be isolated in infected individuals to support this notion.
Unlike wild-type full-length S protein of SARS-CoV-2, the above monomeric fragments do not induce any infection-enhancing antibodies or harmful immune or inflammatory responses , , all of which could be potentially avoided through structure-based immunogen design to improve immunogenicity , However, wide-type full-length or soluble ectodomain form of the SARS-CoV-2 S protein could trigger stronger cellular immune responses , which have been demonstrated to play an important role in controlling diseases caused by CoVs , , including SARS-CoV-2 , and are probably also an important determinant of effective vaccines against SARS-CoV-2 , Genetic variation has been used by many viruses that have RNA genomes , including HIV and influenza, as a mechanism to avoid antibody-mediated immunity, and is partially responsible for the great difficulty in developing effective and durable vaccines against these viruses Furthermore, assays using both monoclonal and polyclonal antibodies generated from individuals naturally infected with D or Gcarrying viruses demonstrated that the DG mutation retains or even increases viral susceptibility to neutralization , , , This suggests that the DG mutant maintains or favors an open, functional conformational state Although at an extremely low frequency, natural variations, including LR AV, VA, and FL that render the S glycoprotein resistant to certain neutralizing antibodies targeting the RBD, emerged under no selection pressure exerted by approved vaccines or neutralizing antibodies or entry inhibitors , However, it has been shown that SARS-CoV-2 escape mutants could be easily selected and quickly amplified under the selection pressure of single antibody treatment These observations suggest that a combination of at least two neutralizing antibodies that recognize and bind to distinct and non-overlapping epitopes on the SARS-CoV-2 S glycoprotein e.
When these observations are taken into consideration for vaccine design and development, an ideal SARS-CoV-2 immunogen should contain as many exposed neutralizing epitopes as possible, although the RBD also possesses extra epitope s besides the epitope in the RBM region 72 , — A safe and efficacious vaccine represents one of the best ways to reduce or eliminate the COVID pandemic Unfortunately, no vaccines for any of the known human CoVs have been licensed , , although several potential SARS-CoV and MERS-CoV vaccines have advanced into human clinical trials for years , , suggesting the development of effective vaccines against human CoVs has always been challenging.
Moreover, a growing number of neutralizing monoclonal antibodies targeting the SARS-CoV-2 S glycoprotein with high potency have been isolated from plenty of convalescent donors 33 as well as humanized mice , , some of which have been shown to afford protection against SARS-CoV-2 challenge in animal models.
It thus seems that vaccine candidates designed to elicit such neutralizing antibodies are feasible. However, it is likely that the S protein has evolved to perform its functions while evading host neutralizing antibody responses and thus should be engineered to ensure an optimal immune response , All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We would like to thank Prof. Xinqi Liu for critical reading of the manuscript; and Drs. Front Public Health Front Immunol Nat Microbiol — Lancet Infect Dis —4. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol — Harrison SC. Viral membrane fusion. Virology — Coronavirus membrane fusion mechanism offers a potential target for antiviral development. Antiviral Res Nat Commun Mechanisms of coronavirus cell entry mediated by the viral spike protein.
Viruses — Cell — Amanat F, Krammer F. In hydrazinolysis, anhydrous hydrazine is added to a salt-free, lyophilized glycoprotein to start the hydrolysis reaction. The released glycan moiety remains intact, whereas the protein may be degraded. Following their release from the glycoprotein, free glycans are usually found in a solution that contains salts, detergents, proteins, peptides, amino acids, and so forth. Before further analysis, these contaminants must be removed.
There are several methods for glycan desalting and purification. The graphitized carbon desalting method is based on the ability of oligosaccharides to bind to carbon beads, while unbound simple monosaccharides, salts, and detergents can be washed away with water [ 39 ]. The glycan moiety is eluted from the beads with acetonitrile ACN , while strongly bound proteins and peptides remain attached.
In another desalting method, a drop of glycan sample is loaded onto a cation-exchange Nafion membrane, which is floated on water. Proteins, peptides, and salts within the sample will bind to the Nafion, and the purified glycan sample can be retrieved [ 40 ]. Similarly to the method with the Nafion membrane, the cation-exchange AG resin can be used. Salts and some detergents may also be removed by dialysis [ 41 ]. Following purification, two approaches may be taken for glycan structure analysis: chromatography and MS.
The fluorescent molecule is conjugated to the glycan reducing end via reductive amination [ 42 ]. However, aminopyridine-based fluorophores can hinder specific glycan characterization, since their conjugation may cause desialylation [ 42 ], whereas the other two fluorophores, 2-AA and 2-AB, may cause only mild desialylation.
The negative charge of 2-AA makes it less appropriate for chromatography and MS, but more suitable for electrophoresis [ 42 ]. Although all methods are efficient, one should carefully choose the appropriate method in the light of the specific requirements and the specific properties of the particular glycans being analyzed.
WAX and gel filtration chromatography require large amounts of oligosaccharides. HPAEC-PAD requires high-pH and high-salt buffers which must be removed before further analysis of the separated glycans, such as in the case of exoglycosidase sequencing. NP-HPLC, having high resolution and reproducibility, is the most efficient method for glycan analysis. In this method, a partially hydrolyzed 2-AB-labeled dextran is used as an external standard, and the retention time of the unknown glycan relative to the standard is converted to glucose units GU [ 43 ].
This GU value is compared to a database of experimental values to obtain a preliminary structure assignment for the glycan [ 46 , 47 ]. Further validation of the glycan sequence can be performed with an array of exoglycosidases, that is, the sequential application of specific exoglycosidases to cleave terminal monosaccharides from the nonreducing end of the glycoprotein [ 49 ].
This chromatographic method also allows relative glycan quantification: by calculating the peak area one can assess the percentage of a specific type of oligosaccharide out of the total glycan repertoire.
Furthermore, relative quantification of the same oligosaccharide in several samples can be achieved [ 43 ]. Several matrices are applicable for glycans, with 2,5-dihydroxybenzoic acid DHB being the most widely used. Yet, in the light of the variety of glycans and their different polarities, 2,4,6-trihydroxyacetophenone THAP is a more suitable matrix [ 50 ]. In ESI and nano-spray, the analyte in solution is converted to an aerosol by the electrospray.
Here the ions are usually multiply charged, which may complicate the glycan analysis [ 51 ]. In MS, the ionization polarity mode should be considered. Although most proteomics and glycomics analyses are performed in a positive-ion mode, the glycan moieties composition is diverse, and if the particular moiety contains N -acetylation and acidic residues, such as sialic acid, ionization may be prevented.
The above notwithstanding, positive ionization can be improved by adopting one of the following two strategies. The glycan moiety can be desialylated by sialidase that cleaves the terminal sialic acid [ 50 ]. Although desialylation enables better ionization, some information is lost: in a pool of released oligosaccharides it is practically impossible to identify which oligosaccharide possessed the sialic acid. Alternatively, permethylation, in which all the glycan-free OH groups are methylated [ 52 , 53 ], will stabilize oligosaccharides containing sialic acid residues.
Moreover, permethylation masks highly polar groups and confers a slight hydrophobicity; thus, the oligosaccharides are more easily separated from contaminants e. Indeed, fragment ions obtained from permethylated derivatives are easily assignable to glycan sequences [ 55 ].
Glycan fragmentation is achieved by cleaving either the glycosidic bonds between monosaccharides or the bonds within a monosaccharide ring termed cross-ring cleavage. The ions obtained from the reducing side are designated X in cross-ring cleavage and Y and Z in glycosidic cleavage.
The nonreducing side ions are designated A in cross-ring cleavage and B and C in glycosidic cleavage Figure 2 [ 41 ]. In collision-induced dissociation CID fragmentation, ions enter a collision cell filled with a gas usually argon and are subjected to high- or low-energy collisions. When performed with high-collision energy, CID results mostly in cross-ring cleavages, providing data on the monosaccharide linkages, while low-collision energy yields glycosidic cleavages, providing data on the monosaccharide sequence and branching [ 56 ].
Recently, high-energy C-trap dissociation HCD fragmentation was applied for glycan and glycoprotein analysis [ 57 ]. The resolution of this method in the low-mass spectrum is higher than that of CID, enabling the identification of the monosaccharide masses. Thus, all the fragment ions, obtained by a combination of techniques, are assigned, providing the full glycan sequence [ 57 , 58 ]. For detailed structural characterization, multiple ion isolation and fragmentation cycles are needed.
For that ion-trap analysis with tandem MS fragmentation is the method of choice as described by Prien et al. Although MALDI and ESI spectra can be used for the elucidation of glycan composition and structure, full glycan characterization by MS is rather complicated: glycan quantity limitations are common, and more importantly, the isobaric structure of many monosaccharides i. While glycan fragmentation may contribute some information on the glycan sequence, branching, and linkage, the combination of several methods is usually necessary to produce a highly reliable picture.
For example, data acquired by MS and exoglycosidase array will give a near-complete characterization of the glycan sequence, branching, and linkage [ 60 ]. Similarly, oligosaccharide hydrolysis after permethylation, followed by gas-chromatography mass-spectrometry GC-MS analysis would identify unmethylated carbons of the monosaccharide. Moreover, structural characterization and branching can be best resolved by NMR analysis if enough material in sufficient purity is available.
Thus, information on branching and inner oligosaccharide linkages could be obtained [ 53 ]. Identification of a glycosylation site is important, since such knowledge could provide an indication of the function of that glycan. As mentioned above, removal of an entire N -glycan moiety by PNGase F or A results in the conversion of Asn to Asp and a shift of one mass unit for each N -glycosylation site. Glycan site mapping is not always straightforward, and some challenges may be encountered.
One challenge is to identify a glycopeptide in a large pool with an absolute majority of nonglycosylated peptides. Moreover, the glycan moiety tends to suppress ionization compared to unmodified peptides.
Therefore, in such a case glycopeptide enrichment should be employed. A vast variety of commercial resin-bound lectins are available for column chromatography [ 63 ]. Another challenge involves the analysis of modified peptides, specifically glycopeptides; in this case, the commonly used CID and HCD fragmentation methods result in intensive fragmentation of the glycan moiety, while the peptide remains intact.
Two recently developed fragmentation methods, electron capture dissociation ECD and electron transfer dissociation ETD , may solve this problem. Both these methods, based on electronic excitation energy, result in peptide fragmentation that leaves the modification intact and attached to the amino acid. A study on N -glycan site mapping by HCD was recently published [ 57 ]: a linear ion-trap orbitrap hybrid mass spectrometer LTQ-orbitrap was used to sequence a glycopeptide through repeated fragmentation MS n.
In the first round of fragmentation MS 2 , CID was used for analyzing glycan sequencing and branching, and HCD analyzed in the orbitrap was used—due to its ability to form glycan oxonium ions—to detect glycopeptides. The Y1 intensity in the HCD spectra is high, and the ion can thus be subjected to further MS 3 for peptide sequencing [ 57 ]. The newly released GlypID 2. The obtained CID fragment ion represents a loss of 69 2-ap instead of 87 for serine, while the mass of threonine and its derivative 2-ab remains This method enables rapid identification of O -GlcNAc-modified peptides in a complex mixture, as well as its site mapping at the low picomole level.
The substoichiometric relation between glycosylation and the protein constitutes a further challenge in glycoprotein quantification. Yet, lectin purification, followed by a labeling procedure, can be used for relative quantification of a specific glycoprotein. The simplest method is lectin blotting see the previous , followed by densitometry of the lectin cross-reactive band.
In addition, lectin chromatography can be used for comparative quantification of a specific glycoprotein in several samples; the relative amounts of a particular glycoprotein, purified by a specific lectin column, in different samples, are determined by densitometry of SDS-PAGE protein bands, stained with either Coomassie blue or another specific stain.
Chromatography by NP-HPLC has previously been used for relative quantification of 2-AB-labeled oligosaccharides in one or more samples [ 43 ]; calculation of the area under the chromatographic peaks indicates the amount of the specific glycan. In addition, chromatography and MS are often coupled to provide a reproducible and reliable glycan characterization and quantification.
MS is also applicable for the relative quantitation of glycopeptides by state-of-the-art methods based on stable isotope labeling, for example, isotope-coded affinity tagging ICAT [ 67 ] or isobaric tags for relative and absolute quantitation iTRAQ [ 68 ].
In these methods, lectin-purified glycoproteins from different samples are each labeled with either light or heavy isotopes during trypsin digestion, mixed together, and analyzed by MS. The relative abundance of the light- and heavy-isotopic peaks of the same peptide from the two different samples indicates their relative quantities. In case of microheterogeneity, that is, the existence of several glycoforms Figure 1 , quantification by a modified permethylation procedure, using isotopic labeling, is applicable [ 54 ].
Following glycan release, each free glycan sample is labeled with either 12 C- or 13 C-methyl iodide; the two samples are mixed in equal proportions, and the labeled glycans are analyzed by MS. This method has a high-dynamic range, adequate linearity, and high reproducibility [ 54 ]. Glycosylation is fundamental for protein function and for cell physiology.
Analysis of the structure and localization of protein glycans is necessary in life sciences and biotechnology. The most reliable analytical tools currently available are chromatography and mass spectrometry. To begin with, characterization of glycans by lectin chromatography is the primary method to be used. MS analysis, by either ETD or isotopic labeling, is suitable for glycosylation site mapping. Isotopic labeling is further appropriate for quantification of glycans Figure 3.
The authors would like to thank Ms. Inez Mureinik for styling the manuscript. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Article of the Year Award: Outstanding research contributions of , as selected by our Chief Editors.
Read the winning articles. Special Issues. Academic Editor: Margarita Stoytcheva. Received 25 Dec Accepted 23 Jan Published 05 Apr Abstract Glycosylation is one of the most abundant posttranslation modifications of proteins, and accumulating evidence indicate that the vast majority of proteins in eukaryotes are glycosylated.
Protein Glycosylation Carbohydrates are essential for cell metabolism and energy production and are building blocks of the extracellular matrix. Figure 1. Basic core structures of N -glycans a and O -glycans b. Table 1. Figure 2. Expected fragment type and mass are indicated in the oligosaccharide structure b. Figure 3. Schematic representation of glycoprotein identification, glycan structural elucidation, and protein glycosylation site mapping and quantification.
References A. View at: Google Scholar E.
0コメント