#2000 Analysis between site-specific N-glycosylation of monoclonal immunoglobulin and complement system activation

Abstract Background and Aims Monoclonal immunoglobulin produced by clonal plasma cells is the main cause in multiple myeloma (MM) and monoclonal gammopathy of renal significance (MGRS). Hypocomplementemia and deposition of complement components in the kidney is observed in some patients with MM or MGRS. Previous studies have suggested that glycosylation of the immunoglobulin was associated with complement-mediated-effector activities. Whereas, site-specific N-glycosylation characterization for monoclonal immunoglobulin in MM or MGRS and its effects on complement system activation still remains unclear. Method We separated plasma monoclonal IgG1 and IgA immunoglobulin from patients with MM and human plasma immunoglobulin from healthy controls (HC). Purified IgG and IgA molecules were digested by trypsin. Tryptic peptides without enrichment of intact N-glycopeptides were analyzed using a combination of electron-transfer/higher-energy collisional dissociation (EThcD) and stepped collision energy/higher-energy collisional dissociation (sceHCD) mass spectrometry (EThcD-sceHCD-MS/MS). N-glycosylation characterization was compared between monoclonal immunoglobulin from MM patients and healthy controls and its association with complement level was evaluated. Results A total of 46 IgG1 digestion samples (HC, n = 16; MM, n = 30) and 38 IgA1 digestion samples (HC, n = 16; MM, n = 22) were analyzed using the EThcDsceHCD-MS/MS. On average, 34 and 37 unique N-glycopeptides of IgG1, 19 unique N-glycans per IgG were identified from HC and MM. The relative abundance of fucosylated N-glycans of IgG1 in MM was significantly higher than HC (94.7% vs 88.8%, p = 0.002). Other types of N-glycans including sialylated, hybrid and complex were similar between the two groups. The types of N-glycans including fucosylated, sialylated, hybrid and complex were similar between patients with hypocomplementemia and normal complement levels. Regarding IgA1, on average, 63 and 52 unique N-glycopeptides of IgA1, 40 and 35 unique N-glycans per IgA were identified from HC and MM. The relative abundance of fucosylated (41.0% vs 26.4%, p < 0.001), sialylated (61.0% vs 45.6%, p = 0.003), mannose (2.44% vs 0.28%, p < 0.001) N-glycans of IgA1 in MM was significantly higher than HC. In MM, the relative abundance of mannose N-glycans of IgA1 in patients with hypocomplementemia was significantly lower than patients with normal complement levels (1.69% vs 7.52%, p < 0.001). The relative abundance of fucosylated and sialylated N-glycans were similar between patients with hypocomplementemia and normal complement levels. Conclusion Site-specific N-glycosylation characterization of monoclonal IgG1 and IgA1 in MM patients were differentially expressed from healthy controls. Certain specific N-glycan types were associated with complement levels. These findings laid a foundation for future investigations of their biological effects.