Language：Japanese   Publisher：生化学工業株式会社  

DOI： 10.32285/glycoforum.27a4j

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

Glycosylation is one of the most important post-translational modifications of cell surface proteins involved in the proliferation, metastasis and treatment resistance of cancer cells. However, little is known about the role of glycosylation as the mechanism of breast cancer cell resistance to endocrine therapy. Herein, we aimed to identify the glycan profiles of tamoxifen-resistant human breast cancer cells, and their potential as predictive biomarkers for endocrine therapy. We established tamoxifen-resistant cells from estrogen receptor-positive human breast cancer cell lines, and their membrane-associated proteins were subjected to lectin microarray analysis. To confirm differential lectin binding to cellular glycoproteins, we performed lectin blotting analyses after electrophoretic separation of the glycoproteins. Mass spectrometry of the tryptic peptides of the lectin-bound glycoproteins was further conducted to identify glycoproteins binding to the above lectins. Finally, expression of the glycans that were recognized by a lectin was investigated using clinical samples from patients who received tamoxifen treatment after curative surgery. Lectin microarray analysis revealed that the membrane fractions of tamoxifen-resistant breast cancer cells showed increased binding to Wisteria floribunda agglutinin (WFA) compared to tamoxifen-sensitive cells. Glycoproteins seemed to be responsible for the differential WFA binding and the results of mass spectrometry revealed several membrane glycoproteins, such as CD166 and integrin beta-1, as candidates contributing to increased WFA binding. In clinical samples, strong WFA staining was more frequently observed in patients who had developed distant metastasis during tamoxifen treatment compared with non-relapsed patients. Therefore, glycans recognized by WFA are potentially useful as predictive markers to identify the tamoxifen-resistant and relapse-prone subset of estrogen receptor-positive breast cancer patients.

DOI： 10.1371/journal.pone.0273513

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/s41388-022-02434-3

PubMed

researchmap

Other Link： https://www.nature.com/articles/s41388-022-02434-3

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

Isohemagglutinin assays employing red blood cells (RBCs) are the most common assays used to measure antibody titer in ABO-incompatible kidney transplantation (ABOi KTx). However, ABO antigens expressed on RBCs are not identical to those of kidney and antibody titers do not always correlate with clinical outcome. We previously reported that CD31 was the main protein linked to ABO antigens on kidney endothelial cells (KECs), which was different from those on RBCs. We developed a new method to measure antibody titer using a microarray of recombinant CD31 (rCD31) linked to ABO antigens (CD31-ABO microarray). Mass spectrometry analysis suggested that rCD31 and native CD31 purified from human kidney had similar ABO glycan. To confirm clinical use of CD31-ABO microarray, a total of 252 plasma samples including volunteers, hemodialysis patients, and transplant recipients were examined. In transplant recipients, any initial IgG or IgM antibody intensity >30,000 against the donor blood type in the CD31-ABO microarray showed higher sensitivity, specificity, positive predictive value, and negative predictive value of AABMR, compared to isohemagglutinin assays. Use of a CD31-ABO microarray to determine antibody titer specifically against ABO antigens expressed on KECs will contribute to precisely predicting AABMR or preventing over immunosuppression following ABOi KTx.

DOI： 10.3389/ti.2022.10248

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Wisteria floribunda agglutinin (WFA)-reactive ceruloplasmin (CP) is a candidate marker for ovarian clear cell carcinoma (CCC) reported in our previous paper. Herein, a new measurement system was developed to investigate its potential as a serum marker for CCC. Site-specific glycome analysis using liquid chromatography/mass spectrometry showed that WFA-CP from CCC binds to WFA via the GalNAcβ1,4GlcNAc (LDN) structure. We used mutant recombinant WFA (rWFA), which has a high specificity to the LDN structure, instead of native WFA, to increase the specificity of the serum sample measurement. To improve the sensitivity, we used a surface plasmon field-enhanced fluorescence spectroscopy immunoassay system, which is approximately 100 times more sensitive than the conventional sandwich enzyme-linked immunosorbent assay system. With these two improvements, the specificity and sensitivity of the serum rWFA-CP measurement were dramatically improved, clearly distinguishing CCC from endometrioma, from which CCC originates. This rWFA-CP assay can be used clinically for the serodiagnosis of early-stage CCC, which is difficult to detect with existing serum markers.

DOI： 10.1021/acs.analchem.1c04302

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

BACKGROUND: Hepatitis B virus (HBV), which causes hepatitis, liver cirrhosis, and hepatocellular carcinoma, is a global human health problem. HBV contains three envelope proteins, S-, M-, and L-hepatitis B surface antigen (HBsAg). We recently found that O-glycosylated M-HBsAg, reactive with jacalin lectin, is one of the primary components of HBV DNA-containing virus particles. Thus, we aimed to analyze and target the glycosylation of HBsAg. METHODS: HBsAg prepared from the serum of Japanese patients with HBV were analyzed using mass spectrometry. The glycopeptide modified with O-glycan was generated and used for immunization. The specificity of the generated antibody and the HBV infection inhibition activity was examined. RESULTS: Mass spectrometry analysis revealed that T37 and/or T38 on M-HBsAg of genotype C were modulated by ±NeuAc(α2,3)Gal(β1,3)GalNAc. Chemically and enzymatically synthesized O-glycosylated peptide (Glyco-PS2) induced antibodies that recognize mainly PreS2 in M-HBsAg not in L-HBsAg, whereas the non-glycosylated peptide (PS2) induced antisera recognizing L-HBsAg but not O-glycosylated M-HBsAg. The removal of O-glycan from M-HBsAg partly decreased the reactivity of the Glyco-PS2 antibody, suggesting that peptide part was also recognized by the antibody. The antibody further demonstrated the inhibition of HBV infection in human hepatic cells in vitro. CONCLUSIONS: Glycosylation of HBsAg occurs differently in different HBsAgs in a site-specific manner. The new Glyco-PS2 antibody, recognizing O-glycosylated M-HBsAg of genotype C, could inhibit HBV infection. GENERAL SIGNIFICANCE: The detailed analysis of HBsAg identified different glycosylations of HBV surface. The glycosylated peptide based on mass spectrometry analysis showed higher potential to induce functional antibody against HBV.

DOI： 10.1016/j.bbagen.2021.130020

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Cypridina noctiluca luciferase (CLuc) is a secreted luminescent protein that reacts with its substrate (Cypridina luciferin) to emit light. CLuc is known to be a thermostable protein and has been used for various research applications, including in vivo imaging and high-throughput reporter assays. Previously, we produced a large amount of recombinant CLuc for crystallographic analysis. However, this recombinant protein did not crystallize, probably due to heterogeneous N-glycan modifications. In this study, we produced recombinant CLuc without glycan modifications by introducing mutations at the N-glycan modification residues using mammalian Expi293F cells, silkworms, and tobacco Bright Yellow-2 cells. Interestingly, recombinant CLuc production depended heavily on the expression hosts. Among these selected hosts, we found that Expi293F cells efficiently produced the recombinant mutant CLuc without significant effects on its luciferase activity. We confirmed the lack of N-glycan modifications for this mutant protein by mass spectrometry analysis but found slight O-glycan modifications that we estimated were about 2% of the ion chromatogram peak area for the detected peptide fragments. Moreover, by using CLuc deletion mutants during the investigation of O-glycan modifications, we identified amino acid residues important to the luciferase activity of CLuc. Our results provide invaluable information related to CLuc function and pave the way for its crystallographic analysis.

DOI： 10.3389/fbioe.2022.774786

PubMed

researchmap

Language：English  

PubMed

Language：English  

PubMed

Language：English  

PubMed

Language：English  

PubMed