High-mannose Glycoform Research Articles

Quantification of the Resilience and Vulnerability of HIV-1 Native Glycan Shield at Atomistic Detail.

SummaryDense surface glycosylation on the HIV-1 envelope (Env) protein acts as a shield from the adaptive immune system. However, the molecular complexity and flexibility of glycans make experimental studies a challenge. Here we have integrated high-throughput atomistic modeling of fully glycosylated HIV-1 Env with graph theory to capture immunologically important features of the shield topology. This is the first complete all-atom model of HIV-1 Env SOSIP glycan shield that includes both oligomannose and complex glycans, providing physiologically relevant insights of the glycan shield. This integrated approach including quantitative comparison with cryo-electron microscopy data provides hitherto unexplored details of the native shield architecture and its difference from the high-mannose glycoform. We have also derived a measure to quantify the shielding effect over the antigenic protein surface that defines regions of relative vulnerability and resilience of the shield and can be harnessed for rational immunogen design.

Quantification of the Resilience and Vulnerability of HIV-1 Native Glycan Shield at Atomistic Detail

Dense surface glycosylation on the HIV-1 envelope (Env) protein acts as a shield from the adaptive immune system. However, the molecular complexity and flexibility of glycans make experimental studies a challenge. Here we have integrated high-throughput atomistic modeling of fully glycosylated HIV-1 Env with graph theory to capture immunologically important features of the shield topology. This is the first complete all-atom model of HIV-1 Env SOSIP glycan shield that includes both oligomannose and complex glycans, providing results which are physiologically more relevant than the previous models with uniform glycosylation. This integrated approach including quantitative comparison with cryo-electron microscopy data provides hitherto unexplored details of the native shield architecture and its difference from the high-mannose glycoform. We have also derived a measure to quantify the shielding effect over the antigenic protein surface that defines regions of relative vulnerability and resilience of the shield and can be harnessed for rational immunogen design.

Lectin AtGAL1 interacts with high-mannose glycoform of the purple acid phosphatase AtPAP26 secreted by phosphate-starved Arabidopsis.

AbstractAmong 29 predicted Arabidopsis purple acid phosphatases (PAPs), AtPAP26 functions as the principle extracellular and intracellular PAP isozyme that is upregulated to recycle and scavenge Pi during Pi‐deprivation or leaf senescence. Our companion paper documented the copurification of a secreted, high‐mannose AtPAP26‐S2 glycoform with AtGAL1 (At1g78850), a Pi starvation‐inducible (PSI), and Galanthus nivalis agglutinin‐related (mannose‐binding) and apple domain lectin. This study tests the hypothesis that AtGAL1 binds AtPAP26‐S2 to modify its enzymatic properties. Far‐western immunodot blotting established that AtGAL1 readily associates with AtPAP26‐S2 but not the low mannose AtPAP26‐S1 glycoform nor other secreted PSI PAPs (i.e., AtPAP12 or AtPAP25). Analytical gel filtration indicated that 55‐kDa AtGAL1 and AtPAP26‐S2 polypeptides associate to form a 112‐kDa heterodimer. Microscopic imaging of transiently expressed, fluorescent protein‐tagged AtGAL1, and associated bimolecular fluorescence complementation assays demonstrated that (a) like AtPAP26, AtGAL1 also localizes to lytic vacuoles of Pi‐deprived Arabidopsis and (b) both proteins interact in vivo. AtGAL1 preincubation significantly enhanced the acid phosphatase activity and thermal stability of AtPAP26‐S2 but not AtPAP26‐S1. We hypothesize that AtGAL1 plays an important role during Pi deprivation through its interaction with mannose‐rich glycans of AtPAP26‐S2 and consequent positive impact on AtPAP26‐S2 activity and stability.

Structural characterization of the Man5 glycoform of human IgG3 Fc

Immunoglobulin G (IgG) consists of four subclasses in humans: IgG1, IgG2, IgG3 and IgG4, which are highly conserved but have unique differences that result in subclass-specific effector functions. Though IgG1 is the most extensively studied IgG subclass, study of other subclasses is important to understand overall immune function and for development of new therapeutics. When compared to IgG1, IgG3 exhibits a similar binding profile to Fcγ receptors and stronger activation of complement. All IgG subclasses are glycosylated at N297, which is required for Fcγ receptor and C1q complement binding as well as maintaining optimal Fc conformation. We have determined the crystal structure of homogenously glycosylated human IgG3 Fc with a GlcNAc2Man5 (Man5) high mannose glycoform at 1.8Å resolution and compared its structural features with published structures from the other IgG subclasses. Although the overall structure of IgG3 Fc is similar to that of other subclasses, some structural perturbations based on sequence differences were revealed. For instance, the presence of R435 in IgG3 (and H435 in the other IgG subclasses) has been implicated to result in IgG3-specific properties related to binding to protein A, protein G and the neonatal Fc receptor (FcRn). The IgG3 Fc structure helps to explain some of these differences. Additionally, protein-glycan contacts observed in the crystal structure appear to correlate with IgG3 affinity for Fcγ receptors as shown by binding studies with IgG3 Fc glycoforms. Finally, this IgG3 Fc structure provides a template for further studies aimed at engineering the Fc for specific gain of function.

Effects of N-Glycan Composition on Structure and Dynamics of IgG1 Fc and Their Implications for Antibody Engineering

Immunoglobulin G1 (IgG1), a subclass of human serum antibodies, is the most widely used scaffold for developing monoclonal antibodies to treat human diseases. The composition of asparagine(N)297-linked glycans can modulate the binding affinity of IgG1 Fc to Fc γ receptors, but it is unclear how the structural modifications of N-glycan termini, which are distal from the binding interface, contribute to the affinity. Through atomistic molecular dynamics simulations of a series of sequentially truncated high-mannose IgG1 Fc glycoforms, we found that the C′E loop and the Cγ2-Cγ3 orientation are highly dynamic, and changes in N-glycan composition alter their conformational ensembles. High-mannose glycoform preferentially samples conformations that are more competent to FcγRIIIa binding, compared to the truncated glycoforms, suggesting a role of IgG1 Fc N-glycan in optimizing the interface with the Fc receptor for efficient binding. The trajectory analyses also reveal that the N-glycan has large amplitude motions and the carbohydrate moiety interconverts between Fc-bound and unbound forms, enabling enzymatic modification of the glycan termini.

BacMam production of active recombinant lecithin–cholesterol acyltransferase: Expression, purification and characterization

Lecithin–cholesterol acyltransferase (LCAT) is a key enzyme in the esterification of cholesterol and its subsequent incorporation into the core of high density lipoprotein (HDL) particles. It is also involved in reverse cholesterol transport (RCT), the mechanism by which cholesterol is removed from peripheral cells and transported to the liver for excretion. These processes are involved in the development of atherosclerosis and coronary heart disease (CHD) and may have therapeutic implications. This work describes the use of baculovirus as a transducing vector to express LCAT in mammalian cells, expression of the recombinant protein as a high-mannose glycoform suitable for deglycosylation by Endo H and its purification to homogeneity and characterization. The importance of producing underglycosylated forms of secreted glycoproteins to obtain high-resolution crystal structures is discussed.

Manganese increases high mannose glycoform on monoclonal antibody expressed in CHO when glucose is absent or limiting: Implications for use of alternate sugars

Alternate sugars such as galactose and fructose are metabolized at a slower rate than glucose and result in lower accumulation of lactate. While low lactate accumulation is desirable, we report that complete substitution of glucose with these sugars results in an increase in M5 high mannose glycans. Surprisingly, this increase is much higher when the culture is supplemented with manganese: for example, when cells are cultured with galactose, M5 high mannose glycan content increased from 5% at 1 nM Mn(2+) in the basal medium to 32% with 16 µM Mn(2+) supplementation. When galactose is supplemented with glucose maintained at low concentrations, a small reduction in high mannose glycans is seen. In control cultures with glucose, the high mannose content was however <2% in this range of Mn(2+) concentration. By varying Mn(2+) and glucose supplementation levels, with or without galactose, we systematically demonstrate that Mn(2+) concentration and glucose availability, together, significantly affect the high mannose glycan content. To our knowledge, this is the first report that shows that the effect of Mn(2+) on high mannose glycan content depends on glucose availability. At each Mn(2+) supplementation level evaluated, galactosylation percentages were highest for cultures where galactose was supplemented with glucose at non-limiting concentration.

Role of ICAM‐1 hypoglycosylation in atherosclerotic plaque formation (1003.1)

N‐glycosylation is an ubiquitous post‐translational modification of proteins secreted from cells or expressed on the cell surface. N‐glycan maturation occurs sequentially moving from high mannose to hybrid to complex glycoforms. We recently showed that that during chronic vascular inflammation that underlies atherosclerosis, endothelial cell surface proteins are hypoglycosylated, characterized by increases in high mannose / hybrid glycoforms. Importantly, these epitopes provide ligands for monocyte rolling and adhesion. The protein(s) or adhesion molecule(s) that harbor high mannose epitopes to mediate monocyte rolling and adhesion was determined in this study. Evidence is provided that that a high mannose glycoform of intercellular adhesion molecule‐1 (ICAM‐1), referred to as HM‐ICAM‐1, is produced in activated endothelial cells. HM‐ICAM‐1 formation was also indicated on human atherosclerotic lesions by proximity ligation assay analysis. Moreover, a role for HM‐ICAM‐1 in mediating monocyte rolling and adhesion was demonstrating in cell culture by expressing ICAM‐1 in Cos‐1 cells in the presence of absence of selective inhibitors of N‐glycan processing (kifunensine or swainsonine) which result in high mannose N‐glycan accumulation. Interestingly, at equal surface expression levels, HM‐ICAM‐1 was more effective at binding monocytes than WT (complex glycosylated ICAM‐1). Finally, we present data showing the effects of mutation of all eight individual N‐glycan binding sites on ICAM‐1 and effects on monocyte rolling and adhesion. The assessment of the specific role of each N‐glycan site in ICAM‐1 may lead to future N‐glycan targeted therapies to modulate atherogenic inflammation.Grant Funding Source: Supported in part by by NIH T32 HL007457, Mechanisms of Hypertension and Cardiovascular Disease

Identification of a high-mannose ICAM-1 glycoform: effects of ICAM-1 hypoglycosylation on monocyte adhesion and outside in signaling

Endothelial adhesion molecules are critical effectors of inflammation ensuring coordinated interactions that allow leukocytes to home to sites of injury. These adhesion molecules are often extensively modified posttranslationaly by the addition of N-glycans, but if, or how, these modifications contribute to the protein function remains poorly understood. Herein we show that activated endothelial cells express two distinct N-glycoforms of intercellular adhesion molecule 1 (ICAM-1) that comprise a complex N-glycoform with α-2,6 sialic acid present at relatively high levels and a second, less abundant and previously undescribed high-mannose glycoform (HM-ICAM-1). This novel HM-ICAM-1 glycoform was also detected in human coronary artery specimens and moreover appeared to be the dominant glycoform in vivo. Production of exclusively HM-ICAM-1 in cells by α-mannosidase inhibition increased monocyte rolling and adhesion compared with mature ICAM-1 consistent with high-mannose epitopes providing leukocyte ligands. Cross-linking of ICAM-1 transmits outside-in signals that affect endothelial permeability and survival. Interestingly, cell signaling (assessed using ERK, VE-cadherin, and Akt phosphorylation) was maintained after cross-linking of HM-ICAM-1 compared with mature ICAM-1; however, interactions with the actin cytoskeleton were lost with HM-ICAM-1. These findings suggest that specific ICAM-1 N-glycoforms modulate distinct aspects of the inflammatory response and identify HM-ICAM-1 as a new therapeutic target for controlling leukocyte trafficking and endothelial inflammation.

Production, characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans

The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.

PGY Repeats and N-Glycans Govern the Trafficking of Paranodin and Its Selective Association with Contactin and Neurofascin-155

Formation of nodes of Ranvier requires contact of axons with myelinating glial cells, generating specialized axo-glial subdomains. Caspr/paranodin is required for the formation of septate-like junctions at paranodes, whereas the related caspr2 is essential for the organization of juxtaparanodes. The molecular mechanisms underlying the segregation of these related glycoproteins within distinct complexes are poorly understood. Exit of paranodin from the endoplasmic reticulum (ER) is mediated by its interaction with F3/contactin. Using domain swapping with caspr2, we mapped a motif with Pro-Gly-Tyr repeats (PGY) in the ectodomain of paranodin responsible for its ER retention. Deletion of PGY allows cell surface delivery of paranodin bypassing the calnexin-calreticulin quality control. Conversely, insertion of PGY in caspr2 or NrCAM blocks these proteins in the ER. PGY is a novel type of processing signal that compels chaperoning of paranodin by contactin. Contactin associated with paranodin is expressed at the cell surface with high-mannose N-glycans. Using mutant CHO lines altered in the processing of N-linked carbohydrates, we show that the high-mannose glycoform of contactin strongly binds neurofascin-155, its glial partner at paranodes. Thus, the unconventional processing of paranodin and contactin may determine the selective association of axo-glial complexes at paranodes.

Protein specific N-glycosylation of tyrosinase and tyrosinase-related protein-1 in B16 mouse melanoma cells

Tyrosinase and tyrosinase-related protein-1 (TRP-1) are two melanogenic enzymes that regulate melanin biosynthesis. Both are glycoproteins and belong to the TRP-1 gene family. They share a significant level of sequence similarity in several regions, including the catalytic domain and the potential N-glycosylation sites. We have recently shown that inhibition of the early steps of N-glycan processing in B16F1 cells dramatically affects tyrosinase activity and melanin synthesis. We present here results on N-glycan processing of TRP-1 and tyrosinase and compare the maturation process and activity of both glycoproteins in the presence of inhibitors of the endoplasmic reticulum stages of N-glycosylation. N-glycan analysis reveals that each of these two glycoproteins contains a mixture of high-mannose and sialylated complex N-glycans. However, in contrast to TRP-1, tyrosinase presents a homogeneous high-mannose glycoform, also. In the presence of alpha-glucosidases inhibitors, the maturation of tyrosinase N-glycans is completely inhibited, whereas TRP-1 is still able to acquire some complex glycans, indicating that endomannosidase acts preferentially on the later glycoprotein. In addition, the dopa-oxidase activity of tyrosinase is totally abolished, whereas for TRP-1 it is only partially affected. The results suggest that despite their structural similarity, tyrosinase is more sensitive than TRP-1 to perturbations of early N-glycan processing, in terms of maturation and catalytical activity.