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IgA nephropathy (IgAN) is a common cause of chronic kidney disease and end-stage renal failure, especially in young people. Due to a wide range of clinical outcomes and difficulty in predicting response to immunosuppression, we need to understand why and identify which patients with IgAN will develop progressive renal impairment. A deletion polymorphism affecting the genes encoding the complement factor H-related protein (FHR)-1 and FHR-3 is robustly associated with protection against IgAN. Some FHR proteins, including FHR-1 and FHR-5, antagonize the ability of complement factor H (fH), the major negative regulator of the complement alternative pathway, to inhibit complement activation on surfaces, a process termed fH deregulation. From a large cohort of patients, we demonstrated that plasma FHR-1 and the FHR-1/fH ratio were elevated in IgAN and associated with progressive disease. Plasma FHR-1 negatively correlated with eGFR but remained elevated in patients with IgAN with normal eGFR. Serum FHR5 was slightly elevated in IgAN but did not correlate with eGFR. Neither FHR5 levels nor the FHR-5/fH ratio was associated with progressive disease. However, higher serum FHR-5 levels were associated with a lack of response to immunosuppression, the presence of endocapillary hypercellularity, and histology scores of disease severity (the Oxford Classification MEST score). Thus, FHR-1 and FHR-5 have a role in IgAN disease progression.

Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 μM DXR P < 0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P < 0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer.

Altered protein glycosylation compared with the disease-free state is a universal feature of cancer cells. It has long been established that distinct glycan structures are associated with specific forms of cancer, but far less is known about the complete array of glycans associated with certain tumors. The cancer glycome has great potential as a source of biomarkers, but progress in this field has been hindered by a lack of available techniques for the elucidation of disease-associated glycosylation. In the present study, lectin microarrays consisting of 45 lectins with different binding preferences covering N- and O-linked glycans were coupled with evanescent-field activated fluorescent detection in the glycomic analysis of primary breast tumors and the serum and urine of patients with metastatic breast cancer. A single 50 µm section of a primary breast tumor or <1 µL of breast cancer patient serum or urine was sufficient to detect glycosylation alterations associated with metastatic breast cancer, as inferred from lectin-binding patterns. The high-throughput, sensitive and relatively simple nature of the simultaneous analysis of N- and O-linked glycosylation following minimal sample preparation and without the need for protein deglycosylation makes the lectin microarray analysis described a valuable tool for discovery phase glycomic profiling.

IntroductionIgA nephropathy (IgAN) is characterized by glomerular deposition of galactose-deficient IgA1 and complement proteins and leads to renal impairment. Complement deposition through the alternative and lectin activation pathways is associated with renal injury.MethodsTo elucidate the contribution of the lectin pathway to IgAN, we measured the 11 plasma lectin pathway components in a well-characterized cohort of patients with IgAN.ResultsM-ficolin, L-ficolin, mannan-binding lectin (MBL)–associated serine protease (MASP)-1 and MBL-associated protein (MAp) 19 were increased, whereas plasma MASP-3 levels were decreased in patients with IgAN compared with healthy controls. Progressive disease was associated with low plasma MASP-3 levels and increased glomerular staining for C3b/iC3b/C3c, C3d, C4d, C5b-9, and factor H–related protein 5 (FHR5). Glomerular FHR5 deposition positively correlated with glomerular C3b/iC3b/C3c, C3d, and C5b-9 deposition, but not with glomerular C4d. These observations, together with the finding that glomerular factor H (fH) deposition was reduced in progressive disease, are consistent with a role for fH deregulation by FHR5 in renal injury in IgAN.ConclusionOur data indicate that circulating MASP-3 levels could be used as a biomarker of disease severity in IgAN and that glomerular staining for FHR5 could both indicate alternative complement pathway activation and be a tissue marker of disease severity.

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Background: Immunoglobulin A (IgA) nephropathy (IgAN) is the most common primary glomerular disease worldwide, and is a leading cause of renal failure. The disease mechanisms are not completely understood, but a higher abundance of galactose-deficient IgA is recognized to play a crucial role in IgAN pathogenesis. While both types of human IgA (IgA1 and IgA2) have several N-glycans as posttranslational modification, only IgA1 features extensive hinge-region O-glycosylation. IgA1 galactose-deficiency on the O-glycans is commonly detected by a lectin-based method. To date, limited detail is known about IgA O- and N-glycosylation in IgAN. Methods: To gain insights into the complex O- and N-glycosylation of serum IgA1 and IgA2 in IgAN, we employed liquid chromatography-mass spectrometry (LC-MS) for the analysis of tryptic glycopeptides of serum IgA from 83 IgAN patients and 244 age and sex-matched healthy controls. Results: Multiple structural features of N-glycosylation of IgA1 and IgA2 were associated with IgAN and glomerular function in our cross-sectional study. These features included differences in galactosylation, sialylation, bisection, fucosylation, and N-glycan complexity. Moreover, IgA1 O-glycan sialylation was associated with both disease risk and glomerular function. Finally, glycopeptides were a better predictor of IgAN and glomerular function than galactose-deficient IgA1 levels measured by lectin-based ELISA. Conclusions: Our high-resolution data suggest that IgA O- and N-glycopeptides are promising targets for future investigations on the pathophysiology of IgAN and as potential noninvasive biomarkers for disease prediction and deteriorating kidney function.

Introduction: Therapeutic agents that target complement are increasingly available for glomerular diseases. However, the mechanisms linking glomerular complement deposition with inflammation and damage are incompletely understood. Complement factor H-related protein 5 (FHR5) interacts with complement C3 and is considered to promote activation. Circulating and glomerular FHR5 associates with IgA nephropathy and abnormal FHR5 associates with familial C3 glomerulopathy (C3G). We characterized glomerular FHR5 staining in C3G and assessed its relationships with histological features of glomerular injury and clinical outcome. Methods:We developed FHR5 staining protocols for formalin-fixed paraffin-embedded (FFPE) renal tissue and applied them to surplus biopsy sections from a C3G cohort.Results: Glomerular FHR5 was highly prevalent in native and transplant C3G and correlated with glomerular C3 and C5b-9 staining. Glomerular FHR5 staining correlated negatively with estimated glomerular filtration rate (eGFR) (P ¼ 0.04, difference of medians 19.7 ml/min per 1.73 m 2 ; 95% confidence interval [CI] 1.1-43.0) and positively with a membranoproliferative glomerulonephritis pattern at diagnostic biopsy (odds ratio 18; 95% CI 1.6-201; P ¼ 0.049). Glomerular FHR5 staining intensity positively correlated with glomerular complement C3b/iC3b/C3c (Pearson's correlation coefficient [R] ¼ 0.59; P ¼ 0.0008), C3dg (R ¼ 0.47; P ¼ 0.02) and C5b9 (R ¼ 0.44, P ¼ 0.02).Conclusions: Glomerular FHR5 is highly prevalent in C3G, interacts with glomerular C3, and is associated with markers of disease severity. Glomerular FHR5 likely exacerbates complement-mediated glomerular damage in C3G and its interaction with glomerular complement might be exploited to target complement therapeutic agents.