Characterization of C3 in C3 glomerulopathy

Sanjeev M Sethi, Julie A. Vrana, Fernando Custodio Fervenza, Jason D. Theis, Amit Sethi, Paul J. Kurtin, Yuzhou Zhang, Richard J H Smith

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Background: C3 glomerulopathy (C3G) is caused by overactivity of the alternative pathway of complement that results in bright glomerular C3 staining with minimal or no deposition of immunoglobulins on immunofluorescence microscopy. Laser microdissection and mass spectrometry of the two subtypes, C3 glomerulonephritis (C3GN) and dense deposit disease (DDD), have identified C3 as the predominant glomerular complement protein, although lesser amounts of C9, C5, C6, C7 and C8 are detectable. C3 plays a central role in complement activity, with its proteolytic cleavage first generating C3a and C3b, followed by inactivation of C3b generating iC3b (which includes C3α and C3β), which undergoes further breakdown yielding C3c and terminal breakdown fragment C3dg. The composition of C3 breakdown products in C3G is not known. Methods: In this study, we chose six cases each of C3GN and DDD to analyze the composition of C3 deposits. We analyzed the amino acid sequence of C3 spectra detected by mass spectrometry to determine the relative abundance of C3 fragments in C3G. Thus we were able to determine the amino acid sequences mapping to the various C3 activation products including C3dg, C3α (C3α1 and α2), and C3β that are part of C3b/iC3b/C3c. Results: C3dg is the predominant cleavage product detected with the highest amino acid coverage. The remaining amino acids map to C3α (C3α1 and α2) and C3β. Amino acids mapping to C3a and C3f are absent. Taken together, the C3α and C3β amino acids represent iC3b prior to or after C3c cleavage of C3dg. The C3 spectra for both C3GN and DDD are surprisingly similar. Conclusion: The finding of large amounts of C3dg suggests that C3b deposition in the glomerulus is an active process triggered by thioester binding of C3b to the glycocalyx overlying the glomerular endothelial cells and glomerular basement membrane. Regulatory protein-mediated inactivation of C3b results in the generation of iC3b. After additional cleavages, mostly C3dg remains.

Original languageEnglish (US)
Pages (from-to)459-465
Number of pages7
JournalNephrology Dialysis Transplantation
Volume32
Issue number3
DOIs
StatePublished - Mar 1 2017

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Complement C3b
Membranoproliferative Glomerulonephritis
Glomerulonephritis
Amino Acids
Amino Acid Sequence
Mass Spectrometry
Alternative Complement Pathway
Glycocalyx
Microdissection
Glomerular Basement Membrane
Fluorescence Microscopy
complement C3d,g
Immunoglobulins
Complement System Proteins
Lasers
Endothelial Cells
Staining and Labeling
Proteins

Keywords

  • C3 glomerulonephritis
  • C3 glomerulopathy
  • C3dg
  • complement
  • dense deposit disease

ASJC Scopus subject areas

  • Nephrology
  • Transplantation

Cite this

Sethi, S. M., Vrana, J. A., Fervenza, F. C., Theis, J. D., Sethi, A., Kurtin, P. J., ... Smith, R. J. H. (2017). Characterization of C3 in C3 glomerulopathy. Nephrology Dialysis Transplantation, 32(3), 459-465. https://doi.org/10.1093/ndt/gfw290

Characterization of C3 in C3 glomerulopathy. / Sethi, Sanjeev M; Vrana, Julie A.; Fervenza, Fernando Custodio; Theis, Jason D.; Sethi, Amit; Kurtin, Paul J.; Zhang, Yuzhou; Smith, Richard J H.

In: Nephrology Dialysis Transplantation, Vol. 32, No. 3, 01.03.2017, p. 459-465.

Research output: Contribution to journalArticle

Sethi, SM, Vrana, JA, Fervenza, FC, Theis, JD, Sethi, A, Kurtin, PJ, Zhang, Y & Smith, RJH 2017, 'Characterization of C3 in C3 glomerulopathy', Nephrology Dialysis Transplantation, vol. 32, no. 3, pp. 459-465. https://doi.org/10.1093/ndt/gfw290
Sethi, Sanjeev M ; Vrana, Julie A. ; Fervenza, Fernando Custodio ; Theis, Jason D. ; Sethi, Amit ; Kurtin, Paul J. ; Zhang, Yuzhou ; Smith, Richard J H. / Characterization of C3 in C3 glomerulopathy. In: Nephrology Dialysis Transplantation. 2017 ; Vol. 32, No. 3. pp. 459-465.
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abstract = "Background: C3 glomerulopathy (C3G) is caused by overactivity of the alternative pathway of complement that results in bright glomerular C3 staining with minimal or no deposition of immunoglobulins on immunofluorescence microscopy. Laser microdissection and mass spectrometry of the two subtypes, C3 glomerulonephritis (C3GN) and dense deposit disease (DDD), have identified C3 as the predominant glomerular complement protein, although lesser amounts of C9, C5, C6, C7 and C8 are detectable. C3 plays a central role in complement activity, with its proteolytic cleavage first generating C3a and C3b, followed by inactivation of C3b generating iC3b (which includes C3α and C3β), which undergoes further breakdown yielding C3c and terminal breakdown fragment C3dg. The composition of C3 breakdown products in C3G is not known. Methods: In this study, we chose six cases each of C3GN and DDD to analyze the composition of C3 deposits. We analyzed the amino acid sequence of C3 spectra detected by mass spectrometry to determine the relative abundance of C3 fragments in C3G. Thus we were able to determine the amino acid sequences mapping to the various C3 activation products including C3dg, C3α (C3α1 and α2), and C3β that are part of C3b/iC3b/C3c. Results: C3dg is the predominant cleavage product detected with the highest amino acid coverage. The remaining amino acids map to C3α (C3α1 and α2) and C3β. Amino acids mapping to C3a and C3f are absent. Taken together, the C3α and C3β amino acids represent iC3b prior to or after C3c cleavage of C3dg. The C3 spectra for both C3GN and DDD are surprisingly similar. Conclusion: The finding of large amounts of C3dg suggests that C3b deposition in the glomerulus is an active process triggered by thioester binding of C3b to the glycocalyx overlying the glomerular endothelial cells and glomerular basement membrane. Regulatory protein-mediated inactivation of C3b results in the generation of iC3b. After additional cleavages, mostly C3dg remains.",
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AU - Sethi, Amit

AU - Kurtin, Paul J.

AU - Zhang, Yuzhou

AU - Smith, Richard J H

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N2 - Background: C3 glomerulopathy (C3G) is caused by overactivity of the alternative pathway of complement that results in bright glomerular C3 staining with minimal or no deposition of immunoglobulins on immunofluorescence microscopy. Laser microdissection and mass spectrometry of the two subtypes, C3 glomerulonephritis (C3GN) and dense deposit disease (DDD), have identified C3 as the predominant glomerular complement protein, although lesser amounts of C9, C5, C6, C7 and C8 are detectable. C3 plays a central role in complement activity, with its proteolytic cleavage first generating C3a and C3b, followed by inactivation of C3b generating iC3b (which includes C3α and C3β), which undergoes further breakdown yielding C3c and terminal breakdown fragment C3dg. The composition of C3 breakdown products in C3G is not known. Methods: In this study, we chose six cases each of C3GN and DDD to analyze the composition of C3 deposits. We analyzed the amino acid sequence of C3 spectra detected by mass spectrometry to determine the relative abundance of C3 fragments in C3G. Thus we were able to determine the amino acid sequences mapping to the various C3 activation products including C3dg, C3α (C3α1 and α2), and C3β that are part of C3b/iC3b/C3c. Results: C3dg is the predominant cleavage product detected with the highest amino acid coverage. The remaining amino acids map to C3α (C3α1 and α2) and C3β. Amino acids mapping to C3a and C3f are absent. Taken together, the C3α and C3β amino acids represent iC3b prior to or after C3c cleavage of C3dg. The C3 spectra for both C3GN and DDD are surprisingly similar. Conclusion: The finding of large amounts of C3dg suggests that C3b deposition in the glomerulus is an active process triggered by thioester binding of C3b to the glycocalyx overlying the glomerular endothelial cells and glomerular basement membrane. Regulatory protein-mediated inactivation of C3b results in the generation of iC3b. After additional cleavages, mostly C3dg remains.

AB - Background: C3 glomerulopathy (C3G) is caused by overactivity of the alternative pathway of complement that results in bright glomerular C3 staining with minimal or no deposition of immunoglobulins on immunofluorescence microscopy. Laser microdissection and mass spectrometry of the two subtypes, C3 glomerulonephritis (C3GN) and dense deposit disease (DDD), have identified C3 as the predominant glomerular complement protein, although lesser amounts of C9, C5, C6, C7 and C8 are detectable. C3 plays a central role in complement activity, with its proteolytic cleavage first generating C3a and C3b, followed by inactivation of C3b generating iC3b (which includes C3α and C3β), which undergoes further breakdown yielding C3c and terminal breakdown fragment C3dg. The composition of C3 breakdown products in C3G is not known. Methods: In this study, we chose six cases each of C3GN and DDD to analyze the composition of C3 deposits. We analyzed the amino acid sequence of C3 spectra detected by mass spectrometry to determine the relative abundance of C3 fragments in C3G. Thus we were able to determine the amino acid sequences mapping to the various C3 activation products including C3dg, C3α (C3α1 and α2), and C3β that are part of C3b/iC3b/C3c. Results: C3dg is the predominant cleavage product detected with the highest amino acid coverage. The remaining amino acids map to C3α (C3α1 and α2) and C3β. Amino acids mapping to C3a and C3f are absent. Taken together, the C3α and C3β amino acids represent iC3b prior to or after C3c cleavage of C3dg. The C3 spectra for both C3GN and DDD are surprisingly similar. Conclusion: The finding of large amounts of C3dg suggests that C3b deposition in the glomerulus is an active process triggered by thioester binding of C3b to the glycocalyx overlying the glomerular endothelial cells and glomerular basement membrane. Regulatory protein-mediated inactivation of C3b results in the generation of iC3b. After additional cleavages, mostly C3dg remains.

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