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Characterisation of a developmentally related polypeptide with glutelin solubility characteristics from Lupinus albus L.

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Abstract

Proteins from Lupinus albus L. cv. Rio Maior seeds were fractionated according to solubility criteria. Patterns of concanavalin A (ConA)-binding polypeptides from the different classes, albumins, globulins, glutelins and prolamins, were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands of apparent molecular masses of 29 and 23.5 kDa with glutelin solubility characteristics bound the lectin. The 23.5-kDa band was separated by two-dimensional electrophoresis into two components: one glycosylated and heterogeneous with an isoelectric point of approx. 10 (designated as G23) and another, not detected with ConA, precipitating in the first dimension. The amino acid and hexosamine analysis of G23 showed that it is particularly rich in Gly (11.2%), Glx (10.0%), Ser (9.0%), Leu (8.2%), Asx (7.5%), and Pro (6.7%) and that it has a considerable content of the sulphur-containing amino acids Met (2.0%) and Cys (5.8%) and contains glucosamine. The determined N-terminal amino acid sequence of G23 was: 1KG(R)V5KGTGD10(T)PXXV15XLY(N)R20T, and this had no significant similarity to any of the amino acid sequences contained in the data bank SWISS-PROT 26. The glycoprotein G23 was completely deglycosylated with peptide-N-glycosidase F, yielding a homogeneous 21-kDa polypeptide composed of approximately 191 amino acids. The structures of the major N-linked neutral oligosaccharides of G23, determined by exoglycosidase sequencing, were as follows: Manα2Manα6(Manα3) Manα6(Manα2Manα2Manα3)Manβ4GlcNAcβ4GlcNAc (13%); ± Manα2Manα6(Manα3)Manα6(± Manα2 Manα2 Manα3)Manβ4GlcNAcβ4GlcNAc (29%); Manα6(Manα3) Manα6(Manα2Manα3)Manβ4GlcNAcβ4GlcNAc (13%); Manα6(Manα3)Manα6(Manα3)Manβ4GlcNAcβ4GlcNAc (16%); Manα6(Manα3)(Xylβ2)Manβ4GlcNAc β4GlcNAc (28%). Changes in G23 abundance during seed development, germination and seedling growth were monitored with a specific antibody. The glycoprotein G23 started to accumulate appreciably during seed formation between the 40th and the 50th days after anthesis and was detected following seed imbibition, until the 9th day in cotyledons, the 2nd day in roots and the 4th day in hypocotyls and leaves.

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Abbreviations

ConA:

concanavalin A

Endo H:

endo-N-acetyl-β-d-glucosaminidase H

GlcNAc:

N-acetylglucosamine

gu:

glucose unit

IEF:

isoelectric focusing

Man:

mannose

NEPHGE:

non-equilibrium pH gradient electrophoresis

PNGase F:

peptide-N-glycosidase F

PVDF:

polyvinylidenedifluoride

Xyl:

xylose

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Author notes

  1. David A. Ashford

    Present address: The Plant Laboratory, Department of Biology, University of York, P.O. Box 373, YO1 5YW, York, UK

Authors and Affiliations

  1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande, 6, Apart. 127, 2780, Oeiras, Portugal

    Júlia Costa & Cândido P. Pinto Ricardo

  2. Instituto de Biologia Experimental e Tecnológica, Apart. 12, 2780, Oeiras, Portugal

    Júlia Costa

  3. Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU, Oxford, UK

    David A. Ashford

  4. Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, 1399, Lisboa Codex, Portugal

    Cândido P. Pinto Ricardo

Authors
  1. Júlia Costa
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  3. Cândido P. Pinto Ricardo
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Additional information

We thank Geoffrey Guile (Oxford Glycobiology Institute, Oxford, UK) for help with HPLC separations and amino acid and hexosamine analysis, Terry Butters (Oxford Glycobiology Institute) for providing the exoglycosidases and advice in their use, Manuela Regala (Instituto de Tecnologia Química e Biológica Oeiras, Portugal) and Paula Veríssimo (University of Coimbra, Portugal) for determining the N-terminal amino acid sequence of G20 and G23 and Dr. Jorge Lampreia (Universidade Nova de Lisboa, Lisbon, Portugal) for the computerised search of the SWISS-PROT data bank. Lupinus albus seeds were provided by Dr. João Neves Martins (Instituto Superior de Agronomia Lisbon, Portugal). We also thank J. Romão (Instituto Gulbenkian de Ciência, Oeiras, Portugal) for technical assistance in antibody production. This work was supported by Junta National de Investigação Científica e Tecnológica, Portugal.

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Costa, J., Ashford, D.A. & Pinto Ricardo, C.P. Characterisation of a developmentally related polypeptide with glutelin solubility characteristics from Lupinus albus L.. Planta 198, 221–229 (1996). https://doi.org/10.1007/BF00206247

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  • DOI: https://doi.org/10.1007/BF00206247

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