Elimination of Potential Sites of Glycosylation Fails to Abrogate Complement Regulatory Function of Cell Surface CD59

Rother, Russell P.; Zhao, Jinmin; Zhou, Quansheng; Sims, P.

doi:10.1074/jbc.271.39.23842

Cited by 16 publications

(7 citation statements)

References 23 publications

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“…A UPAR mutant lacking all of the N-glycosylation sites remained trapped in the ER and did not reach the cell surface (5). In the case of CD59, however, N-glycosylation did not appear to be crucial, inasmuch as eliminating the N-glycosylation site did not reduce the cell-surface expression of the molecule or adversely affect its function (12).…”

Section: Discussionmentioning

confidence: 95%

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Glycosylation of Asn-76 in mouse GPIHBP1 is critical for its appearance on the cell surface and the binding of chylomicrons and lipoprotein lipase

Beigneux

Gin

Davies

et al. 2008

Journal of Lipid Research

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GPIHBP1 is a glycosylphosphatidylinositolanchored protein in the lymphocyte antigen 6 (Ly-6) family that recently was identified as a platform for the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1 binds both LPL and chylomicrons and is expressed on the luminal face of microvascular endothelial cells. Here, we show that mouse GPIHBP1 is N-glycosylated at Asn-76 within the Ly-6 domain. Human GPIHBP1 is also glycosylated. The N-linked glycan could be released from mouse GPIHBP1 with N-glycosidase F, endoglycosidase H, or endoglycosidase F1. The glycan was marginally sensitive to endoglycosidase F2 digestion but resistant to endoglycosidase F3 digestion, suggesting that the glycan on GPIHBP1 is of the oligomannose type. Mutating the N-glycosylation site in mouse GPIHBP1 results in an accumulation of GPIHBP1 in the endoplasmic reticulum and a markedly reduced amount of the protein on the cell surface. Consistent with this finding, cells expressing a nonglycosylated GPIHBP1 lack the ability to bind LPL or chylomicrons. Eliminating the N-glycosylation site in a truncated soluble version of GPIHBP1 causes a modest reduction in the secretion of the protein. These studies demonstrate that N-glycosylation of GPIHBP1 is important for the trafficking of GPIHBP1 to the cell surface.-Beigneux, A. P., P.

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Section: Discussionmentioning

confidence: 95%

“…Several GPI-anchored proteins containing Ly-6 domains, such as UPAR and CD59, are known to be N-glycosylated (5,6,12,13). In the current study, we examined the idea Fig.…”

Section: Discussionmentioning

confidence: 99%

Glycosylation of Asn-76 in mouse GPIHBP1 is critical for its appearance on the cell surface and the binding of chylomicrons and lipoprotein lipase

Beigneux

Gin

Davies

et al. 2008

Journal of Lipid Research

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“…1A), the matched CD59 spots in the comparison of PDAC, CP and healthy samples were poorly resolved, forming a vertical streak. This may be due to heterogeneity in added carbohydrates [39] that may have masked any significant changes in abundance in the subsequent experiment. Neither of two antibodies used for validation detected the 14 kDa fragment seen on our 2-D gels, possibly indicating the need for a specific antibody that would target this specific fragment in urine.…”

Section: Discussionmentioning

confidence: 99%

Analysis of the urine proteome in patients with pancreatic ductal adenocarcinoma

Weeks

Hariharan

Petronijevic³

et al. 2008

Proteomics Clinical Apps

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Pancreatic ductal adenocarcinoma (PDAC) accounts for over 213 000 deaths worldwide each year, largely due to late diagnosis. One of the risk factors for the development of PDAC is chronic pancreatitis (CP); the intense desmoplastic reaction makes differentiation between the two conditions extremely difficult. In order to identify biomarkers for noninvasive diagnosis, we performed 2-D DIGE analysis of urine samples from healthy individuals and patients with PDAC and CP. Despite considerable intersample heterogeneity, a total of 127 statistically valid (p<0.05), differentially expressed protein spots were detected, 101 of which were identified using MALDI-TOF MS. A number of these, including annexin A2, gelsolin and CD59 have already been associated with PDAC, however, their validation using immunoblotting proved challenging. This is probably due to extensive PTMs and processing thus indicating the need for raising specific antibodies for urinary proteins. Despite this, our study clearly demonstrates that urine is a valid source of noninvasive biomarkers in patients with pancreatic diseases.

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“…[9,10,21] Just as carbohydrates may not be required for THP’s cofactor activity, the N-glycans present on other complement regulatory proteins, such as C4-binding protein, decay accelerating factor (DAF, CD55) and CD59 are not required for their complement inhibitory activity. [40,41,42]…”

Section: Discussionmentioning

confidence: 99%

Human Tamm-Horsfall protein, a renal specific protein, serves as a cofactor in complement 3b degradation

Rhodes

2017

PLoS ONE

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Tamm-Horsfall protein (THP) is an abundant urinary protein of renal origin. We hypothesize that THP can act as an inhibitor of complement since THP binds complement 1q (C1q) of the classical complement pathway, inhibits activation of this pathway, and is important in decreasing renal ischemia-reperfusion injury (a complement-mediated condition). In this study, we began to investigate whether THP interacted with the alternate complement pathway via complement factor H (CFH). THP was shown to bind CFH using ligand blots and in an ELISA (KD of 1 × 10−6 M). Next, the ability of THP to alter CFH’s normal action as it functioned as a cofactor in complement factor I (CFI)–mediated complement 3b (C3b) degradation was investigated. Unexpectedly, control experiments in these in vitro assays suggested that THP, without added CFH, could act as a cofactor in CFI-mediated C3b degradation. This cofactor activity was present equally in THP isolated from 10 different individuals. While an ELISA demonstrated small amounts of CFH contaminating THP samples, these CFH amounts were insufficient to explain the degree of cofactor activity present in THP. An ELISA demonstrated that THP directly bound C3b (KD ~ 5 × 10−8 m), a prerequisite for a protein acting as a C3b degradation cofactor. The cofactor activity of THP likely resides in the protein portion of THP since partially deglycosylated THP still retained cofactor activity. In conclusion, THP appears to participate directly in complement inactivation by its ability to act as a cofactor for C3b degradation, thus adding support to the hypothesis that THP might act as an endogenous urinary tract inhibitor of complement.

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Elimination of Potential Sites of Glycosylation Fails to Abrogate Complement Regulatory Function of Cell Surface CD59

Cited by 16 publications

References 23 publications

Glycosylation of Asn-76 in mouse GPIHBP1 is critical for its appearance on the cell surface and the binding of chylomicrons and lipoprotein lipase

Glycosylation of Asn-76 in mouse GPIHBP1 is critical for its appearance on the cell surface and the binding of chylomicrons and lipoprotein lipase

Analysis of the urine proteome in patients with pancreatic ductal adenocarcinoma

Human Tamm-Horsfall protein, a renal specific protein, serves as a cofactor in complement 3b degradation

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