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N-glycan moieties of the crustacean egg yolk protein and their glycosylation sites

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Abstract

Vitellogenin (Vg) is the precursor of the egg yolk glycoprotein of crustaceans. In the prawn Macrobrachium rosenbergii, Vg is synthesized in the hepatopancreas, secreted to the hemolymph, and taken up by means of receptor-mediated endocytosis into the oocytes. The importance of glycosylation of Vg lies in its putative role in the folding, processing and transport of this protein to the egg yolk and in the fact that the N-glycan moieties could provide a source of carbohydrate during embryogenesis. The present study describes, for the first time, the structure of the glycan moieties and their sites of attachment to the Vg of M. rosenbergii. Bioinformatics analysis revealed seven putative N-glycosylation sites in M. rosenbergii Vg; two of these glycosylation sites are conserved throughout the Vgs of decapod crustaceans from the Pleocyemata suborder (N 159 and N 660). The glycosylation of six putative sites of M. rosenbergii Vg (N 151, N 159, N 168, N 614, N 660 and N 2300) was confirmed; three of the confirmed glycosylation sites are localized around the N-terminally conserved N-glycosylation site N 159. From a theoretical three-dimensional structure, these three N-glycosylated sites N 151, N 159, and N 168 were localized on the surface of the Vg consensus sequence. In addition, an uncommon high mannose N-linked oligosaccharide structure with a glucose cap (Glc1Man9GlcNAc2) was characterized in the secreted Vg. These findings thus make a significant contribution to the structural elucidating of the crustacean Vg glycan moieties, which may shed light on their role in protein folding and transport and in recognition between Vg and its target organ, the oocyte.

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Abbreviations

ACN:

acetonitrile

DIG:

digoxigenin

DSA:

Datura stramonium agglutinin

DTT:

dithiothreitol

ER:

endoplasmic reticulum

Glc:

glucose

GlcNAc:

N-acetylglucosamine

GII:

glucosidase II

GNA:

Galanthus nevallis agglutinin

GU:

glucose unit

HDL:

high density lipoprotein

Hex:

hexose

HexNAc:

N-acetylhexosamine

HPLC:

high performance liquid chromatography

JBM:

jack bean α-mannosidase

MAA:

Maackia amurensis agglutinin

Man:

mannose

MALDI:

matrix-assisted laser desorption/ionization

PNA:

peanut (Arachis hypogaea) agglutinin

PNGase F:

peptide-N-glycosidase F

SDS-PAGE:

sodium dodecyl sulfate polyacrylamide gel electrophoresis

SNA:

Sambucus nigra agglutinin

TFA:

trifluoroacetic acid

Vg:

vitellogenin

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Acknowledgments

We thank Ayana Benet-Perlberg and the team from the Dor experimental station of the Ministry of Agriculture for providing animal culture facilities, Mr. Tomer Ventura for his technical assistance and Ms. Lilah Glazer for her help in generating the 3D model. This work was supported by an internal grant from Ben Gurion University of the Negev; I. K. is the incumbent of MAOF fellowship from the Israel Council for Higher Education, Planning and Budgeting Committee.

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Authors and Affiliations

  1. Department of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O.Box 653, Beer-Sheva, 84105, Israel

    Ziv Roth & Isam Khalaila

  2. Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel

    Ziv Roth, Shmuel Parnes, Simy Wiel & Amir Sagi

  3. The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel

    Ziv Roth, Shmuel Parnes, Simy Wiel & Amir Sagi

  4. Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD, 21202, USA

    Nili Zmora & J. Sook Chung

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  1. Ziv Roth
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  2. Shmuel Parnes
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  7. Isam Khalaila
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Correspondence to Isam Khalaila.

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Roth, Z., Parnes, S., Wiel, S. et al. N-glycan moieties of the crustacean egg yolk protein and their glycosylation sites. Glycoconj J 27, 159–169 (2010). https://doi.org/10.1007/s10719-009-9268-3

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