Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

Yang, Chenxi; Park, Arick C.; Davis, Nancy D.; Russell, John N.; Kim, Byoung Jae; Brand, David; Lawrence, Matthew; Ge, Ying; Westphall, Michael S.; Coon, Joshua J.; Greenspan, Daniel S.

doi:10.1074/jbc.m112.406850

Cited by 46 publications

(78 citation statements)

References 81 publications

(96 reference statements)

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“…The observation that Col V also stimulated IL-1β and TNFα in isolated monocytes in a P2X7R dependent manner, is consistent with the idea that Col V, a heavily post transcriptionally modified protein (23), can activate leukocytes directly as previously reported for lung epithelial cells in culture (37). How this occurs, and whether Pattern Recognition Receptors (PRRs) are involved is unclear.…”

Section: Discussionsupporting

confidence: 90%

“…Therefore, we examined whether P2X7R inhibition blocks the production of IL-1β or TNFα from monocytes in Col V and α1-peptide stimulated whole PBMCs, and as a control for possible T-independent effects, in cultures of isolated monocytes. Furthermore, since the Col V molecule, containing numerous post translational modifications (23) could potentially activate the T cell as well as the antigen presenting cell for Col V, the monocyte, through Damage Associated Molecular Patterns (DAMPs) interactions, we investigated whether Col V and DR-restricted peptides could stimulate monocytes directly by separating monocytes from PBMCs and stimulating isolated monocytes. Figure 4A shows representative flow plots of Col V, p1049 and p1439 stimulated PBMCs, in the presence or absence of Suramin or AZD9056.…”

Section: Resultsmentioning

confidence: 99%

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Differential Requirement for P2X7R Function in IL-17 Dependent vs. IL-17 Independent Cellular Immune Responses

Sullivan

Jankowska‐Gan

Shi

et al. 2014

American Journal of Transplantation

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IL17-dependent autoimmunity to Collagen type V (Col V) has been associated with lung transplant obliterative bronchiolitis. Unlike the Th-1-dependent immune responses to tetanus toxoid (TT), the Th17 response to Col V in lung transplant patients and its Th1/17 variant observed in coronary artery disease patients requires IL-1β, TNFα and CD14+ cells. Given the involvement of the P2X7R in monocyte IL-1β responses, we investigated its role in Th17, Th1/17, and Th1- mediated pro-inflammatory responses. Transfer of antigen-pulsed PBMC from Col V -reactive patients into SCID mouse footpads along with P2X7R antagonists revealed a selective inhibition of Col V-, but not TT -specific swelling responses. P2X7R inhibitors blocked IL-1β induction from monocytes, including both Col V-α1 peptide-induced (T-dependent), as well as native Col V induced (T-independent) responses. Significantly higher P2X7R expression was found on CXCR3negCCR4+/6+ CD4+ [Th17] vs. CXCR3+CCR4/6neg CD4+ [Th1] subsets in PBMC, suggesting that the paradigm of selective dependence on P2X7R might extend beyond Col V autoimmunity. Indeed, P2X7R inhibitors suppressed not only anti-Col V, but also Th1/17–mediated allo-immunity, in a heart transplant patient without affecting anti-viral EBV responses. These results suggest that agents targeting the P2X7R might effectively treat Th17-related transplant pathologies, while maintaining Th1-immunity to infection.

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Section: Discussionsupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Differential Requirement for P2X7R Function in IL-17 Dependent vs. IL-17 Independent Cellular Immune Responses

Sullivan

Jankowska‐Gan

Shi

et al. 2014

American Journal of Transplantation

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“…15 We also searched for HyP residues in the Xaa position of “Gly-Xaa-[Ala/Gln/Val]” motifs, which have been recently identified in other collagens. 59,68 These searches led to the identification of an additional HyP (P 481 ) residue in the Xaa position of the Gly-Xaa-Gln motif. Although the Xaa position of HyP residue suggests that hydroxylation may occur in the third position of the proline ring, there is no previous chemical evidence to support this assignment.…”

Section: Discussionmentioning

confidence: 99%

Comprehensive Characterization of Glycosylation and Hydroxylation of Basement Membrane Collagen IV by High-Resolution Mass Spectrometry

et al. 2015

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Collagen IV is the main structural protein that provides a scaffold for assembly of basement membrane proteins. Posttranslational modifications such as hydroxylation of proline and lysine and glycosylation of lysine are essential for the functioning of collagen IV triple-helical molecules. These modifications are highly abundant posing a difficult challenge for in-depth characterization of collagen IV using conventional proteomics approaches. Herein, we implemented an integrated pipeline combining high-resolution mass spectrometry with different fragmentation techniques and an optimized bioinformatics workflow to study posttranslational modifications in mouse collagen IV. We achieved 82% sequence coverage for the α1 chain, mapping 39 glycosylated hydroxylysine, 148 4-hydroxyproline, and seven 3-hydroxyproline residues. Further, we employed our pipeline to map the modifications on human collagen IV and achieved 85% sequence coverage for the α1 chain, mapping 35 glycosylated hydroxylysine, 163 4-hydroxyproline, and 14 3-hydroxyproline residues. Although lysine glycosylation heterogeneity was observed in both mouse and human, 21 conserved sites were identified. Likewise, five 3-hydroxyproline residues were conserved between mouse and human, suggesting that these modification sites are important for collagen IV function. Collectively, these are the first comprehensive maps of hydroxylation and glycosylation sites in collagen IV, which lay the foundation for dissecting the key role of these modifications in health and disease.

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“…But with isolated monocyte populations only ColV, but not synthetic ColV α1 peptides could induce IL1β and TNFα within the cells. This finding suggests that while ColV peptides can trigger T cells in whole PBMC to activate the monocytes, only ColV with all its tertiary structure and post-translational modifications[39] can provide a Damage Associated Molecular Pattern (DAMP) recognized by isolated monocytes[38]. In addition to P2X7R, we have also found a striking difference in the role of Leukocyte Associated Immunoglobulin-like Receptor 1 (LAIR1) in ColV responses[40].…”

Section: Introductionmentioning

confidence: 99%

Autoimmune Reactivity in Graft Injury: Player or Bystander?

Agashe

Burlingham

2015

Curr Transpl Rep

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Organ transplantation is the only viable treatment for several end-stage organ failures. However chronic rejection prevents long-term graft survival. Traditionally this rejection was attributed to the development of alloimmunity in transplant patients. However recent evidence suggests that autoimmunity plays a larger role in chronic rejection of certain organ transplants, than alloimmunity. In this review we will focus on the history of autoimmunity in solid-organ transplantation and at look the Collagen Type V, K-α-tubulin, Vimentin, Cardiac myosin and Heat Shock Proteins as classical examples of auto-antigens in organ transplantation. We will also look at some of the recent reports looking at the mechanisms of autoimmunity and try to provide answers to some of the age-old questions in autoimmunity.

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Comprehensive Mass Spectrometric Mapping of the Hydroxylated Amino Acid residues of the α1(V) Collagen Chain

Cited by 46 publications

References 81 publications

Differential Requirement for P2X7R Function in IL-17 Dependent vs. IL-17 Independent Cellular Immune Responses

Differential Requirement for P2X7R Function in IL-17 Dependent vs. IL-17 Independent Cellular Immune Responses

Comprehensive Characterization of Glycosylation and Hydroxylation of Basement Membrane Collagen IV by High-Resolution Mass Spectrometry

Autoimmune Reactivity in Graft Injury: Player or Bystander?

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