Similarities and differences in the structures and proteoform profiles of the complement proteins C6 and C7

Lukassen, Marie V.; Franc, Vojtěch; Hevler, Johannes F.; Heck, Albert J. R.

doi:10.1002/pmic.202000310

Cited by 5 publications

(4 citation statements)

References 47 publications

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“…C6 also has a partially occupied C‐mannosylation site at TSR2 residue W69 in a noncanonical GPWSDCDP sequence [11]. This modification was not present in the C‐mannosylated crystal structure in the PDB and C‐mannosylated W69 is the less abundant proteoform (30%) [28]. Nonetheless, if approximately a third of circulating C6 monomers have this modification present, it may still influence the interactions or stability of C6 in the complement system.…”

Section: Resultsmentioning

confidence: 99%

“…Whether the variable occupancy of the C‐mannosylation site on C9 can subtly influence the assembly of C9 monomers, or perhaps their interactions with pathogen membranes, remains to be seen. The heterogeneity of C‐mannosylation on human plasma‐derived C6 has been demonstrated [28] and these data showed that the crystallized proteoform was the most abundant and thus provides a strong representation of C‐mannosylation in soluble, monomeric C6. The variable occupancy of C‐mannosylation sites in the three TSR domains contributes to the overall heterogeneity of soluble C6 and may be particularly influential in the assembled MAC.…”

Section: Resultsmentioning

confidence: 99%

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Molecular basis of C‐mannosylation – a structural perspective

Crine

Acharya

2021

The FEBS Journal

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The structural and functional diversity of proteins can be enhanced by numerous post-translational modifications. C-mannosylation is a rare form of glycosylation consisting of a single alpha or beta Dmannopyranose forming a carbon-carbon bond with the pyrrole ring of a tryptophan residue. Despite first being discovered in 1994, C-mannosylation is still poorly understood and 3D structures are available for only a fraction of the total predicted C-mannosylated proteins. Here we present the first comprehensive review of C-mannosylated protein structures by analysing the data for all ten proteins with C-mannosylation/s deposited in the Research Collaboratory for Structural Bioinformatics Protein Data Bank. We analysed in detail the WXXW/WXXWXXW consensus motif and the highly conserved pair of arginine residues in thrombospondin type-1 repeat Cmannosylation sites or homologous arginine residues in other domains. Furthermore, we identified a conserved PXP sequence C-terminal of the C-mannosylation site. The PXP motif forms a tight turn region in the polypeptide chain and its universal conservation in C-mannosylated protein is worthy of further experimental study. The stabilisation of C-mannopyranosyl groups was demonstrated through hydrogen bonding with arginine and other charged or polar amino acids. Where possible, the structural findings were linked to other functional studies demonstrating the role of Cmannosylation in protein stability, secretion, or function. With the current technological advances in structural biology, we hope to see more progress in the study of C-mannosylation that may correspond to discoveries of novel C-mannosylation pathways and functions with implications for human health and biotechnology.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Molecular basis of C‐mannosylation – a structural perspective

Crine

Acharya

2021

The FEBS Journal

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“…Previous studies have shown that almost all complement proteins can bear sialylated glycans [82][83][84] . Factor H, which inhibits the cascade, binds α2,3 sialylation on host-cells, a critical aspect of self-recognition 85 .…”

Section: Discussionmentioning

confidence: 99%

α2,6-Sialylation is Upregulated in Severe COVID-19 Implicating the Complement Cascade

Qin

Kurz

Chen

et al. 2022

Preprint

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Better understanding of the mechanisms of COVID-19 severity is desperately needed in current times. Although hyper-inflammation drives severe COVID-19, precise mechanisms triggering this cascade and what role glycosylation might play therein is unknown. Here we report the first high-throughput glycomic analysis of COVID-19 plasma samples and autopsy tissues. We find α2,6-sialylation is upregulated in plasma of patients with severe COVID-19 and in the lung. This glycan motif is enriched on members of the complement cascade, which show higher levels of sialylation in severe COVID-19. In the lung tissue, we observe increased complement deposition, associated with elevated α2,6-sialylation levels, corresponding to elevated markers of poor prognosis (IL-6) and fibrotic response. We also observe upregulation of the α2,6-sialylation enzyme ST6GAL1 in patients who succumbed to COVID-19. Our work identifies a heretofore undescribed relationship between sialylation and complement in severe COVID-19, potentially informing future therapeutic development.

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“…nearly obsolete but also decreases the required protein amounts as well as undesired over-length cross-links. In contrast to classical in-solution XL-MS workflows, IGX-MS allows the differentiation of conformation-and interaction-specific distance restraints as it has been shown for various mitochondrial complexes but also for plasma protein complexes, the mitotic checkpoint complex (MCC) as well as for different assemblies of the archaeal ammonia monooxygenase (176)(177)(178)(179). IGX-MS seems specially promising for future modeling studies that aim at characterizing cooccurring protein complexes with different stoichiometries or assembly states.…”

Section: Combining Xl-ms and Cp-ms For The Structural Analysis Of Pro...mentioning

confidence: 99%

Structural Characteristics of Mitochondrial Protein Assemblies Probed by Mass Spectrometry

Hevler¹

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Most of the biological processes that maintain cellular life depend on habitually interacting proteins, which are forming complexes that correlate with the type of biological processes they are involved in. In order to understand these processes, X-ray crystallography, cryogenic electron microscopy/tomography and mass spectrometry are commonly used to structurally characterize macromolecular protein assemblies. As briefly reviewed in Chapter 1, each of these methods has benefits and drawbacks associated, however most of the methods predominantly require a highly purified sample. The downside being that macromolecular protein assemblies are, up to more recently, rarely studied in their naïve cellular environment. In this thesis, the versatility of cross-linking mass-spectrometry (XL-MS), to characterize protein assemblies in vivo is demonstrated. We show that especially in mitochondria, for which cryo-ET identification is limited to the biggest or most "uniquely-shaped" complexes (Bauerlein and Baumeister, 2021) such as the ATP synthase XL-MS enables the sensitive identification of novel mitochondrial protein complexes. Although XL-MS enables the characterization of protein complexes in their naïve cellular environment, established XL-MS workflows present drawbacks that significantly hamper an adequate structural characterization. The presence of coinciding respiratory chain protein assemblies in mitochondria (Wittig et al., 2006) for instance, prevent the confident identification of a cross-links origin and thereby an accurate structural characterization of a specific assembly. To tackle existing challenges of classical in-solution XL-MS, we therefore set out to develop a novel, easy-to use workflow (Chapter 2) termed in-gel cross-linking mass spectrometry (IGX-MS). IGX-MS circumvents time and sample consuming steps, and further provides a sensible solutions for differentiating cross-links obtained from co-occurring protein oligomers or complexes, leading up to an improved structural characterization. Further, by combining XL-MS with complementary structural techniques, e.g. complexome profiling (CP-MS) or cryo-ET enabled us to identify and structurally characterize novel protein complexes in mitochondria (Chapter 3-5). By performing XL-MS and CP-MS for the structural analysis of macromolecular protein assemblies in bovine heart mitochondria (Chapter 3), we uncover that a substantial amount of dimeric apoptosis factor 1 (AIFM1) is associated with at least 10% of monomeric cytochrome c oxidase (COX). In a collaborative effort with the Zeev-Ben-Mordehai group (Utrecht University), we highlight that ambiguity in cryo-ET experiments can be overcome by additionally performing XL-MS experiments (Chapter 4). Using sub-tomographic averaging, we identified that in mammalian sperm, mitochondria are wrapped around the flagellar cytoskeleton, which is mediated through conserved arrays of glycerol kinase-like proteins. Finally yet importantly, in Chapter 5 we describe how XL-MS can be utilized for the characterization of protein complexes using computer-aided structural modeling. Like described in Chapter 3, XL-MS and CP-MS data is used for the detailed characterization of mitochondrial protein assemblies, providing essential information regarding complex members and assembly state prior to structural modeling. Combining data from both methods enabled us to identify MRPS36 as a novel component of the 2-oxoglutarate dehydrogenase complex (OGDHC), and subsequently was utilized to refine the AI-driven structural model. The final model provides new insights into the topology of this multi-component enzyme as well as details on its enzymatic function.

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Similarities and differences in the structures and proteoform profiles of the complement proteins C6 and C7

Cited by 5 publications

References 47 publications

Molecular basis of C‐mannosylation – a structural perspective

Molecular basis of C‐mannosylation – a structural perspective

α2,6-Sialylation is Upregulated in Severe COVID-19 Implicating the Complement Cascade

Structural Characteristics of Mitochondrial Protein Assemblies Probed by Mass Spectrometry

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