Volume 118, Issue 2 pp. 147-152

Original Article

Differential enzymatic deglycosylation reveals attachment of red cell B antigen onto the carbohydrate moiety of glycophorin A and glycophorin B

Kate Hsu,

Corresponding Author

Kate Hsu

[email protected]

orcid.org/0000-0003-1009-0997

The Laboratory of Immunogenetics, Department of Medical Research, MacKay Memorial Hospital, New Taipei City, Taiwan

Department of Nursing, MacKay Junior College of Medicine, Nursing, and Management, New Taipei City, Taiwan

Institute of Biomedical Sciences, MacKay Medical College, New Taipei City, Taiwan

Correspondence

Kate Hsu, MacKay Memorial Hospital, Transfusion Medicine & Immunogenetics Laboratories, 45 Min-Sheng Rd, Research building 616, Tamsui 251, Taiwan.

Email: [email protected]

Search for more papers by this author

Kate Hsu,

Corresponding Author

Kate Hsu

[email protected]

orcid.org/0000-0003-1009-0997

The Laboratory of Immunogenetics, Department of Medical Research, MacKay Memorial Hospital, New Taipei City, Taiwan

Department of Nursing, MacKay Junior College of Medicine, Nursing, and Management, New Taipei City, Taiwan

Institute of Biomedical Sciences, MacKay Medical College, New Taipei City, Taiwan

Correspondence

Kate Hsu, MacKay Memorial Hospital, Transfusion Medicine & Immunogenetics Laboratories, 45 Min-Sheng Rd, Research building 616, Tamsui 251, Taiwan.

Email: [email protected]

Search for more papers by this author

First published: 12 December 2022

https://doi.org/10.1111/vox.13385

Funding information: Mackay Memorial Hospital, Grant/Award Numbers: MMH 111-26, MMH 112-63; Taiwan National Science and Technology Council, Grant/Award Number: MOST 110-2628-B-195-001

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Abstract

Background and Objectives

Early studies indicate that red cell A and B antigens are attached primarily onto band 3 and GLUT1 on the erythrocyte membrane and little onto glycophorin A (GPA) and glycophorin B (GPB). But as GPA and band 3 form stable protein complexes and GPA is much more heavily glycosylated than band 3, this study re-examined the association between ABO antigens and GPA/GPB.

Materials and Methods

Band 3/GPA-associated protein complexes were first immunoprecipitated, followed by differential enzymatic deglycosylation that removed sialic acids, N-glycans and O-glycans. Serological anti-A (BIRMA 1) and anti-B IgM (GAMA 110) could be used for western blot (WB); however, only the anti-B IgM showed significant reactivity for the immunoprecipitates isolated by anti-band 3. The expression of the B antigen in un-deglycosylated and differentially deglycosylated band 3 immunoprecipitates was thus compared.

Results

Besides attachment to band 3, red cell B antigen expressed substantially on GPA monomer and homodimer, GPA*GPB heterodimer, and GPB monomer and dimer via attachments through the N- and O-glycans.

Conclusion

Immunoprecipitation (IP), as a means of protein separation and concentration, was used in combination with a WB to differentiate glycosylation on different proteins and oligomers. This study implemented differential enzymatic deglycosylation during IP of the band 3 complexes. This combined approach allowed separate identification of the B antigen on GPA/GPB monomer and dimer and GPA*GPB heterodimer, and band 3 on the WB and verified non-trivial expression of the B antigen on GPA and GPB on the erythrocyte surface.

CONFLICT OF INTEREST

The author declares no conflict of interests or competing financial interests.

Supporting Information

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Volume118, Issue2

February 2023

Pages 147-152

Differential enzymatic deglycosylation reveals attachment of red cell B antigen onto the carbohydrate moiety of glycophorin A and glycophorin B

Abstract

Background and Objectives

Materials and Methods

Results

Conclusion

CONFLICT OF INTEREST

Supporting Information

REFERENCES

References

Information

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Differential enzymatic deglycosylation reveals attachment of red cell B antigen onto the carbohydrate moiety of glycophorin A and glycophorin B

Abstract

Background and Objectives

Materials and Methods

Results

Conclusion

CONFLICT OF INTEREST

Supporting Information

REFERENCES

References

Related

Information