IgA Nephropathy Caused by Unusual Polymerization of IgA1 with Aberrant N-Glycosylation in a Patient with Monoclonal Immunoglobulin Deposition Disease

<div><p>Immunoglobulin A nephropathy (IgAN) is a form of chronic glomerulonephritis characterized by the deposition of IgA immune complexes in the glomerular region. The cause of IgAN is unknown, but multiple mechanisms have been suggested. We previously reported a rare case of mesangioproliferative glomerulonephritis in a patient with monoclonal immunoglobulin deposition disease associated with monoclonal IgA1. In this study, we performed the detailed analyses of serum IgA1 from this patient in comparison with those from patients with mIgA plasma cell disorder without renal involvement and healthy volunteers. We found unusual polymerization of IgA1 with additional <i>N</i>-glycosylation distinctive in this patient, which was different from known etiologies. Glycan profiling of IgA1 by the lectin microarray revealed an intense signal for <i>Wisteria floribunda</i> agglutinin (WFA). This signal was reduced by disrupting the native conformation of IgA1, suggesting that the distinct glycan profile was reflecting the conformational alteration of IgA1, including the glycan conformation detected as additional <i>N</i>-glycans on both the heavy and light chains. This unusually polymerized state of IgA1 would cause an increase of the binding avidity for lectins. WFA specifically recognized highly polymerized and glycosylated IgA1. Our results of analysis in the rare case of a patient with monoclonal immunoglobulin deposition disease suggest that the formation of unusually polymerized IgA1 is caused by divergent mechanisms including multiple structural alterations of glycans, which contributes to IgA1 deposition and mesangium proliferation.</p></div

Sandwich lectin ELISA of sequential deglycosylated IgA1 in mIgA-MIDD.

<p>IgA1 purified from mIgA-MIDD serum was digested with neuraminidase, and then β-galactosidase or β1,4-galactosidase. Digested and undigested samples were subjected to a sandwich lectin ELISA with HPA (A), VVA (B), PNA (C), and WFA (D) as described in the Methods. The relative intensity of each lectin was normalized to the IgA1 concentration. HPA, <i>Helix pomatia</i> agglutinin; VVA, <i>Vicia villosa</i> lectin; PNA, peanut agglutinin.</p

Differential glycan profiles of purified IgA1 after trypsin digestion.

<p>IgA1 purified from sera of three HVs, two MPCD patients, and one mIgA-MIDD patient was incubated with trypsin. Each tryptic digest was labeled with Cy3-SE and subjected to the lectin microarray. The relative intensities of lectins were normalized to the maximum fluorescence intensity.</p

Differential glycan profiles of purified IgA1.

<p>IgA1 purified from sera of three HVs, two MPCD patients, and one mIgA-MIDD patient was subjected to lectin microarray. IgA1-binding signals on the lectin microarray were detected with a biotinylated anti-IgA1 mAb. The relative intensity of each lectin was normalized to the maximum fluorescence intensity. mIgA-MIDD, monoclonal immunoglobulin deposition disease associated with monoclonal IgA; MPCD, monoclonal IgA plasma cell disorder; HV, healthy volunteers.</p

SDS-agarose gel electrophoresis of purified IgA1.

<p>IgA1 purified from sera of two HV, two MPCD patients, and an mIgA-MIDD patient was incubated in SDS buffer and then subjected to agarose gel electrophoresis analysis.</p

Time course of PNGase F treatment of IgA1.

<p>IgA1 purified from the sera of the mIgA-MIDD patient (A) and an MPCD patient (B) was digested partially with PNGase F for the indicated times (lanes 1–5, 7–10). Complete digestion of <i>N</i>-glycans was performed by incubation with PNGase F for 120 min (lanes 6, 11). Arrowheads indicate the position of PNGase F.</p

Large-Scale Identification of <i>N-</i>Glycan Glycoproteins Carrying Lewis x and Site-Specific <i>N-</i>Glycan Alterations in <i>Fut9</i> Knockout Mice

The Lewis x (Le^x) structure (Galβ1–4­(Fucα1–3)­GlcNAc-R) is a carbohydrate epitope comprising the stage-specific embryonic antigen-1 (SSEA-1) and CD15, and it is synthesized by α1,3-fucosyltransferase 9 (Fut9). <i>Fut9</i> is expressed specifically in the stomach, kidney, brain, and in leukocytes, suggesting a specific function in these tissues. In this study, the <i>N</i>-linked glycan mass spectrometry profile of wild-type mouse kidney glycoproteins revealed the presence of abundant terminal fucoses, which were lost following knockout of the <i>Fut9</i> gene; the terminal fucose was therefore concluded to be Le^x. These results suggested that Le^x presence is widespread rather than being limited to specific proteins. We endeavored to comprehensively identify the Le^x carriers in the mouse kidney. Glycopeptides carrying fucosylated glycans were collected by Aleuria aurantia lectin (AAL) affinity chromatography from kidney homogenates of wild-type and <i>Fut9</i> knockout mice. The site-specific <i>N</i>-glycomes on the glycopeptides were subsequently analyzed by adopting a new glycoproteomic technology composed of dissociation-independent assignment of glycopeptide signals and accurate mass-based prediction of the <i>N</i>-glycome on the glycopeptides. Our analyses demonstrated that 24/32 glycoproteins contained the Le^x <i>N</i>-glycan structure in wild-type kidney; of these, Le^x was lost from 21 in the knockout mice. This is the first report of large-scale identification of Le^x-carrying glycoproteins from a native sample based on the site-specific glycome analysis

Glycobiomarker, Fucosylated Short-Form Secretogranin III Levels Are Increased in Serum of Patients with Small Cell Lung Carcinoma

Secretogranin III (SgIII) is a member of the chromogranin/secretogranin family of neuroendocrine secretory proteins. Granins are expressed in endocrine and neuroendocrine cells and subsequently processed into bioactive hormones. Although granin-derived peptide expression is correlated with neuroendocrine carcinomas, little is known about SgIII. We previously identified SgIII by a comparative glycoproteomics approach for elucidation of glycobiomarker candidates in lung carcinoma. Here, we examined the expression, secretion, and glycosylation of SgIII to identify novel biomarkers of small cell lung carcinoma (SCLC). In comparative immunohistochemical analysis and secretion profiling, SgIII was observed in all types of lung cancer. However, low-molecular-weight SgIII (short-form SgIII) was specifically found in SCLC culture medium. Glycoproteomics analysis showed that a fucosylated glycan was attached to the first of three potential <i>N</i>-glycosylation sites and an unfucosylated glycan was detected on the second site; however, the third site was not glycosylated. Next, we performed lectin capture with a fucose-binding lectin and detected short-form SgIII specifically in the sera of patients with SCLC. The results suggested an association between the fucosylated glycoform of short-form SgIII and SCLC. Thus, fucosylated short-form SgIII may be a valuable biomarker for SCLC and could be used to monitor development of the disease. All MS data are available via ProteomeXchange and jPOST with identifiers PXD007626 and JPST000313, respectively

Glycoproteomics Approach for Identifying Glycobiomarker Candidate Molecules for Tissue Type Classification of Non-small Cell Lung Carcinoma

Histopathological classification of lung cancer has important implications in the application of clinical practice guidelines and the prediction of patient prognosis. Thus, we focused on discovering glycobiomarker candidates to classify the types of lung cancer tissue. First, we performed lectin microarray analysis of lung cancer tissue specimens and cell lines and identified Aleuria aurantia lectin (AAL), <i>Hippeastrum</i> hybrid lectin (HHL), and Concanavalia ensiformis agglutinin (ConA) as lectin probes specific to non-small cell lung carcinoma (NSCLC). LC–MS-based analysis was performed for the comprehensive identification of glycoproteins and N-linked glycosylation sites using lectin affinity capture of NSCLC-specific glycoforms of glycoproteins. This analysis identified 1092 AAL-bound glycoproteins (316 gene symbols) and 948 HHL/ConA-bound glycoproteins (279 gene symbols). The lectin microarray-assisted verification using 15 lung cancer cell lines revealed the NSCLC-specific expression of fibronectin. The glycosylation profiling of fibronectin indicated that the peanut agglutinin (PNA) signal appeared to differentiate two NSCLC types, adenocarcinoma and large cell carcinoma, whereas the protein expression level was similar between these types. Our glycoproteomics approach together with the concurrent use of an antibody and lectin is applicable to the quantitative and qualitative monitoring of variations in glycosylation of fibronectin specific to certain types of lung cancer tissue

Large-scale Identification of <i>N-</i>Glycosylated Proteins of Mouse Tissues and Construction of a Glycoprotein Database, GlycoProtDB

Protein glycosylation is a common post-translational modification that plays important roles in terms of protein function. However, analyzing the relationship between glycosylation and protein function remains technically challenging. This problem arises from the fact that the attached glycans possess diverse and heterogeneous structures. We believe that the first step to elucidate glycan function is to systematically determine the status of protein glycosylation under physiological conditions. Such studies involve analyzing differences in glycan structure on cell type (tissue), sex, and age, as well as changes associated with perturbations as a result of gene knockout of glycan biosynthesis-related enzyme, disease and drug treatment. Therefore, we analyzed a series of glycoproteomes in several mouse tissues to identify glycosylated proteins and their glycosylation sites. Comprehensive analysis was performed by lectin- or HILIC-capture of glycopeptide subsets followed by enzymatic deglycosylation in stable isotope-labeled water (H₂¹⁸O, IGOT) and finally LC–MS analyses. In total, 5060 peptides derived from 2556 glycoproteins were identified. We then constructed a glycoprotein database, GlycoProtDB, using our experimental-based information to facilitate future studies in glycobiology