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Unusual N-type glycosylation of salivary prolactin-inducible protein (PIP): multiple LewisY epitopes generate highly-fucosylated glycan structures

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Abstract

Prolactin-inducible protein (PIP) is a glycoprotein found in body secretions from exocrine glands like saliva and seminal plasma. Important biological functions of PIP concentrations have been demonstrated, e.g. in tumor diagnosis and progression. PIP quantity has been also found useful to determine the success of chemotherapy of mammary carcinoma. Here, we present the analysis of the N-glycosylation of PIP isolated from different sources by LC-MS(/MS) and 1H-NMR. We found a very uncommon N-type glycosylation of PIP in healthy individuals from both, seminal fluid and saliva. PIP carries unusual highly fucosylated N-linked glycans with multiple Lewisy (Ley) epitopes on bi-, tri- and tetraantennary structures resulting in up to nine fucosyl residues on a tetraantennary glycan. In most organs, Ley epitopes are not present on N-glycans except in case of a tumor when it is highly up-regulated and important for prognosis. Here, for the first time on a specific glycoprotein Ley antigens are unambiguously characterized on an N-type glycan by NMR spectroscopy. So far, for specific glycoproteins Ley epitopes had only been reported on O-glycans. Furthermore, a correlation between a nonsynonymous single nucleotide polymorphism (SNP) and glycosylation pattern was detected: individuals heterozygous for the SNP causing the amino acid exchange 51Gln to 51His have glycan structures with a higher degree of sialylation compared to individuals lacking the SNP.

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Abbreviations

ACN:

Acetonitrile

ASD:

Autism Spectrum Disorder

AIEX:

Anion exchange chromatography

CID:

Collision-induced dissociation

DC-SIGN:

Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin

DEAE FF:

Diethylaminoethyl cellulose fast flow

ESI:

Electrospray ionization

ExAC:

Exome aggregation consortium

Gal:

Galactose

GlcNAc:

N-acetylglucosamine

GCDFP-15:

Gross cystic disease fluid protein-15

gp17:

Glycoprotein 17

F:

Deoxyhexose

FA:

Formic acid

Fuc:

Fucose

H:

Hexose

HPLC:

High performance liquid chromatography

HR:

High resolution

Lea/b/x/y :

Lewis antigen a/b/x/y

MAF:

Minor allele frequency

MALDI:

Matrix assisted laser desorption/ionization

Man:

Mannose

MS:

Mass spectrometry

NMR:

Nuclear magnetic resonance spectroscopy

N:

Hexosamine

NSCLC:

Non-small cell lung cancer

PGC:

Porous graphitic carbon

PIP:

Prolactin-inducible protein

PNGase F:

N-glycosidase F

PSA:

Prostate specific antigen

RP:

Reversed phase

S:

Sialic acid

SABP:

Secretory actin-binding protein

SEC:

Size exclusion chromatography

SNP:

Single nucleotide polymorphism

TOCSY:

Total correlated spectroscopy

Tris:

Tris(hydroxymethyl)aminomethane

UV:

Ultraviolet

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Acknowledgments

This work was supported by the Helmholtz Association by a stipend for A.W.

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Author notes

  1. Henning N. Behnken

    Present address: Global Structure Analytics - Crop Protection, BASF SE, APD/ES LI444, 67117, Limburgerhof, Germany

Authors and Affiliations

  1. Department of Chemistry, Institute of Organic Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, 20146, Hamburg, Germany

    Alena Wiegandt, Henning N. Behnken & Bernd Meyer

Authors
  1. Alena Wiegandt
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  2. Henning N. Behnken
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  3. Bernd Meyer
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Contributions

Conceived and designed the experiments: AW HNB BM. Performed the experiments: AW HNB. Analyzed the data: AW HNB BM. Contributed reagents/materials/analysis tools: BM. Wrote the paper: AW BM.

Corresponding author

Correspondence to Bernd Meyer.

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The authors declare that they have no conflict of interest.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Wiegandt, A., Behnken, H.N. & Meyer, B. Unusual N-type glycosylation of salivary prolactin-inducible protein (PIP): multiple LewisY epitopes generate highly-fucosylated glycan structures. Glycoconj J 35, 323–332 (2018). https://doi.org/10.1007/s10719-018-9826-7

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