N-glycosylation influences human corticosteroid-binding globulin measurements

Lesley A. Hill, Zeynep Sumer-Bayraktar, John G. Lewis, Eva Morava-Kozicz, Morten Thaysen-Andersen, Geoffrey L. Hammond

Research output: Contribution to journalArticle

Abstract

Objective: Discrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) using detection monoclonal antibodies that recognize an epitope (9G12) within its reactive center loop (RCL), versus an epitope (12G2) in a different location, have suggested that CBG with a proteolytically cleaved RCL exists in blood samples. We have previously been unable to verify this biochemically, and sought to determine if N-glycosylation differences account for discrepancies in ELISA measurements of CBG. Methods and subjects: Molecular biological, biochemical and glycopeptide analyses were used to examine how N-glycosylation at specific sites, including at N347 within the RCL, affect CBG ELISA or steroid-binding capacity assay (BCA) measurements. Plasma from patients with congenital disorders of glycosylation (CDG) was also examined in these assays as examples of N-glycosylation defects. Results: We demonstrate that an N-glycan at N347 within the CBG RCL limits the 9G12 antibody from recognizing its epitope, whereas the 12G2 antibody reactivity is unaffected, thereby contributing to discrepancies in ELISA measurements using these two antibodies. Qualitative differences in N-glycosylation at N238 also negatively affect the steroid-binding of CBG in the absence of an N-glycan at N347 caused by a T349A substitution. Desialylation increased both ELISA measurements relative to BCA values. Similarly, plasma CBG levels in both ELISAs were much higher than BCA values in several CDG patients. Conclusions: Plasma CBG measurements are influenced by variations in N-glycosylation. This is important given the increasing number of CDG defects identified recently and because N-glycosylation abnormalities are common in patients with metabolic and liver diseases.

Original languageEnglish (US)
Pages (from-to)1136-1148
Number of pages13
JournalEndocrine Connections
Volume8
Issue number8
DOIs
StatePublished - Aug 1 2019

Fingerprint

Transcortin
Glycosylation
Congenital Disorders of Glycosylation
Enzyme-Linked Immunosorbent Assay
Epitopes
Polysaccharides
Antibodies
Steroids
Glycopeptides
Metabolic Diseases
Liver Diseases
Monoclonal Antibodies

Keywords

  • Congenital disorders of glycosylation
  • Epitopes
  • Glycosylation
  • Monoclonal antibodies
  • Protein structure
  • Steroid binding

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism
  • Endocrinology

Cite this

Hill, L. A., Sumer-Bayraktar, Z., Lewis, J. G., Morava-Kozicz, E., Thaysen-Andersen, M., & Hammond, G. L. (2019). N-glycosylation influences human corticosteroid-binding globulin measurements. Endocrine Connections, 8(8), 1136-1148. https://doi.org/10.1530/EC-19-0242

N-glycosylation influences human corticosteroid-binding globulin measurements. / Hill, Lesley A.; Sumer-Bayraktar, Zeynep; Lewis, John G.; Morava-Kozicz, Eva; Thaysen-Andersen, Morten; Hammond, Geoffrey L.

In: Endocrine Connections, Vol. 8, No. 8, 01.08.2019, p. 1136-1148.

Research output: Contribution to journalArticle

Hill, LA, Sumer-Bayraktar, Z, Lewis, JG, Morava-Kozicz, E, Thaysen-Andersen, M & Hammond, GL 2019, 'N-glycosylation influences human corticosteroid-binding globulin measurements', Endocrine Connections, vol. 8, no. 8, pp. 1136-1148. https://doi.org/10.1530/EC-19-0242
Hill LA, Sumer-Bayraktar Z, Lewis JG, Morava-Kozicz E, Thaysen-Andersen M, Hammond GL. N-glycosylation influences human corticosteroid-binding globulin measurements. Endocrine Connections. 2019 Aug 1;8(8):1136-1148. https://doi.org/10.1530/EC-19-0242
Hill, Lesley A. ; Sumer-Bayraktar, Zeynep ; Lewis, John G. ; Morava-Kozicz, Eva ; Thaysen-Andersen, Morten ; Hammond, Geoffrey L. / N-glycosylation influences human corticosteroid-binding globulin measurements. In: Endocrine Connections. 2019 ; Vol. 8, No. 8. pp. 1136-1148.
@article{b72dac3bae9e4c32af1b64035e4c0709,
title = "N-glycosylation influences human corticosteroid-binding globulin measurements",
abstract = "Objective: Discrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) using detection monoclonal antibodies that recognize an epitope (9G12) within its reactive center loop (RCL), versus an epitope (12G2) in a different location, have suggested that CBG with a proteolytically cleaved RCL exists in blood samples. We have previously been unable to verify this biochemically, and sought to determine if N-glycosylation differences account for discrepancies in ELISA measurements of CBG. Methods and subjects: Molecular biological, biochemical and glycopeptide analyses were used to examine how N-glycosylation at specific sites, including at N347 within the RCL, affect CBG ELISA or steroid-binding capacity assay (BCA) measurements. Plasma from patients with congenital disorders of glycosylation (CDG) was also examined in these assays as examples of N-glycosylation defects. Results: We demonstrate that an N-glycan at N347 within the CBG RCL limits the 9G12 antibody from recognizing its epitope, whereas the 12G2 antibody reactivity is unaffected, thereby contributing to discrepancies in ELISA measurements using these two antibodies. Qualitative differences in N-glycosylation at N238 also negatively affect the steroid-binding of CBG in the absence of an N-glycan at N347 caused by a T349A substitution. Desialylation increased both ELISA measurements relative to BCA values. Similarly, plasma CBG levels in both ELISAs were much higher than BCA values in several CDG patients. Conclusions: Plasma CBG measurements are influenced by variations in N-glycosylation. This is important given the increasing number of CDG defects identified recently and because N-glycosylation abnormalities are common in patients with metabolic and liver diseases.",
keywords = "Congenital disorders of glycosylation, Epitopes, Glycosylation, Monoclonal antibodies, Protein structure, Steroid binding",
author = "Hill, {Lesley A.} and Zeynep Sumer-Bayraktar and Lewis, {John G.} and Eva Morava-Kozicz and Morten Thaysen-Andersen and Hammond, {Geoffrey L.}",
year = "2019",
month = "8",
day = "1",
doi = "10.1530/EC-19-0242",
language = "English (US)",
volume = "8",
pages = "1136--1148",
journal = "Endocrine Connections",
issn = "2049-3614",
publisher = "BioScientifica Ltd.",
number = "8",

}

TY - JOUR

T1 - N-glycosylation influences human corticosteroid-binding globulin measurements

AU - Hill, Lesley A.

AU - Sumer-Bayraktar, Zeynep

AU - Lewis, John G.

AU - Morava-Kozicz, Eva

AU - Thaysen-Andersen, Morten

AU - Hammond, Geoffrey L.

PY - 2019/8/1

Y1 - 2019/8/1

N2 - Objective: Discrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) using detection monoclonal antibodies that recognize an epitope (9G12) within its reactive center loop (RCL), versus an epitope (12G2) in a different location, have suggested that CBG with a proteolytically cleaved RCL exists in blood samples. We have previously been unable to verify this biochemically, and sought to determine if N-glycosylation differences account for discrepancies in ELISA measurements of CBG. Methods and subjects: Molecular biological, biochemical and glycopeptide analyses were used to examine how N-glycosylation at specific sites, including at N347 within the RCL, affect CBG ELISA or steroid-binding capacity assay (BCA) measurements. Plasma from patients with congenital disorders of glycosylation (CDG) was also examined in these assays as examples of N-glycosylation defects. Results: We demonstrate that an N-glycan at N347 within the CBG RCL limits the 9G12 antibody from recognizing its epitope, whereas the 12G2 antibody reactivity is unaffected, thereby contributing to discrepancies in ELISA measurements using these two antibodies. Qualitative differences in N-glycosylation at N238 also negatively affect the steroid-binding of CBG in the absence of an N-glycan at N347 caused by a T349A substitution. Desialylation increased both ELISA measurements relative to BCA values. Similarly, plasma CBG levels in both ELISAs were much higher than BCA values in several CDG patients. Conclusions: Plasma CBG measurements are influenced by variations in N-glycosylation. This is important given the increasing number of CDG defects identified recently and because N-glycosylation abnormalities are common in patients with metabolic and liver diseases.

AB - Objective: Discrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) using detection monoclonal antibodies that recognize an epitope (9G12) within its reactive center loop (RCL), versus an epitope (12G2) in a different location, have suggested that CBG with a proteolytically cleaved RCL exists in blood samples. We have previously been unable to verify this biochemically, and sought to determine if N-glycosylation differences account for discrepancies in ELISA measurements of CBG. Methods and subjects: Molecular biological, biochemical and glycopeptide analyses were used to examine how N-glycosylation at specific sites, including at N347 within the RCL, affect CBG ELISA or steroid-binding capacity assay (BCA) measurements. Plasma from patients with congenital disorders of glycosylation (CDG) was also examined in these assays as examples of N-glycosylation defects. Results: We demonstrate that an N-glycan at N347 within the CBG RCL limits the 9G12 antibody from recognizing its epitope, whereas the 12G2 antibody reactivity is unaffected, thereby contributing to discrepancies in ELISA measurements using these two antibodies. Qualitative differences in N-glycosylation at N238 also negatively affect the steroid-binding of CBG in the absence of an N-glycan at N347 caused by a T349A substitution. Desialylation increased both ELISA measurements relative to BCA values. Similarly, plasma CBG levels in both ELISAs were much higher than BCA values in several CDG patients. Conclusions: Plasma CBG measurements are influenced by variations in N-glycosylation. This is important given the increasing number of CDG defects identified recently and because N-glycosylation abnormalities are common in patients with metabolic and liver diseases.

KW - Congenital disorders of glycosylation

KW - Epitopes

KW - Glycosylation

KW - Monoclonal antibodies

KW - Protein structure

KW - Steroid binding

UR - http://www.scopus.com/inward/record.url?scp=85071161372&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85071161372&partnerID=8YFLogxK

U2 - 10.1530/EC-19-0242

DO - 10.1530/EC-19-0242

M3 - Article

AN - SCOPUS:85071161372

VL - 8

SP - 1136

EP - 1148

JO - Endocrine Connections

JF - Endocrine Connections

SN - 2049-3614

IS - 8

ER -