Correct glycosylation, Golgi-processing, and targeting to protein bodies of the vacuolar protein phytohemagglutinin in transgenic tobacco

Arnd Sturm, Toni A. Voelker, Eliot M Herman, Maarten J. Chrispeels

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

We used a heterologous system (transgenic Nicotiana tabacum L.) to investigate the processing, assembly and targeting of phytohemagglutinin (PHA), the lectin of the common bean, Phaseolus vulgaris L. In the bean, this glycoprotein accumulates in the protein bodies of the storage parenchyma cells in the cotyledons, and each polypeptide has a high-mannose glycan attached to Asn12 and a complex glycan on Asn60. The gene for PHA-L, dlec2, with 1200 basepairs (bp) 5′ upstream and 1600 bp 3′ downstream from the coding sequence was introduced into tobacco using Agrobacterium-mediated transformation (T. Voelker et al., 1987, EMBO J. 6, 3571-3577). Examination of thin sections of tobacco seeds by immunocytochemistry with antibodies against PHA showed that PHA-L accumulated in the amorphous matrix of the protein bodies in the embryo and endosperm. This localization was confirmed using a non-aqueous method to isolate the protein bodies from mature tobacco seeds. The biochemical analysis of tobacco PHA indicated that the signal peptide had been correctly removed, and that the polypeptides formed 6.4 S oligomers; tobacco PHA had a high-mannose glycan at Asn12 and a complex glycan at Asn60. The presence of the complex glycan shows that transport to the protein bodies was mediated by the Golgi complex. At seed maturity, a substantial portion of the PHA-L remained associated with the endoplasmic reticulum and the Golgi complex, as indicated by fractionation experiments using aqueous media and the presence of two high-mannose glycans on some of the polypeptides. Taken together, these data show that insertion of the nascent PHA into the endoplasmic reticulum, signal peptide processing, glycosylation, assembly into oligomers, glycan modification in the Golgi, and targeting of the protein occur faithfully in this heterologous system, although transport may not be as efficient as in bean cotyledons.

Original languageEnglish (US)
Pages (from-to)170-183
Number of pages14
JournalPlanta
Volume175
Issue number2
DOIs
StatePublished - Aug 1988
Externally publishedYes

Fingerprint

protein bodies
phytohemagglutinin
Phytohemagglutinins
Protein Transport
glycosylation
Glycosylation
Tobacco
tobacco
Polysaccharides
genetically modified organisms
polysaccharides
Proteins
Mannose
proteins
mannose
polypeptides
Seeds
beans
Cotyledon
Golgi Apparatus

Keywords

  • Golgi apparatus
  • Nicotiana (transgenic)
  • Phaseolus (phytohemagglutinin)
  • Phytohemagglutinin
  • Protein bodies
  • Protein targeting
  • Transformation (tobacco)

ASJC Scopus subject areas

  • Plant Science

Cite this

Correct glycosylation, Golgi-processing, and targeting to protein bodies of the vacuolar protein phytohemagglutinin in transgenic tobacco. / Sturm, Arnd; Voelker, Toni A.; Herman, Eliot M; Chrispeels, Maarten J.

In: Planta, Vol. 175, No. 2, 08.1988, p. 170-183.

Research output: Contribution to journalArticle

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abstract = "We used a heterologous system (transgenic Nicotiana tabacum L.) to investigate the processing, assembly and targeting of phytohemagglutinin (PHA), the lectin of the common bean, Phaseolus vulgaris L. In the bean, this glycoprotein accumulates in the protein bodies of the storage parenchyma cells in the cotyledons, and each polypeptide has a high-mannose glycan attached to Asn12 and a complex glycan on Asn60. The gene for PHA-L, dlec2, with 1200 basepairs (bp) 5′ upstream and 1600 bp 3′ downstream from the coding sequence was introduced into tobacco using Agrobacterium-mediated transformation (T. Voelker et al., 1987, EMBO J. 6, 3571-3577). Examination of thin sections of tobacco seeds by immunocytochemistry with antibodies against PHA showed that PHA-L accumulated in the amorphous matrix of the protein bodies in the embryo and endosperm. This localization was confirmed using a non-aqueous method to isolate the protein bodies from mature tobacco seeds. The biochemical analysis of tobacco PHA indicated that the signal peptide had been correctly removed, and that the polypeptides formed 6.4 S oligomers; tobacco PHA had a high-mannose glycan at Asn12 and a complex glycan at Asn60. The presence of the complex glycan shows that transport to the protein bodies was mediated by the Golgi complex. At seed maturity, a substantial portion of the PHA-L remained associated with the endoplasmic reticulum and the Golgi complex, as indicated by fractionation experiments using aqueous media and the presence of two high-mannose glycans on some of the polypeptides. Taken together, these data show that insertion of the nascent PHA into the endoplasmic reticulum, signal peptide processing, glycosylation, assembly into oligomers, glycan modification in the Golgi, and targeting of the protein occur faithfully in this heterologous system, although transport may not be as efficient as in bean cotyledons.",
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T1 - Correct glycosylation, Golgi-processing, and targeting to protein bodies of the vacuolar protein phytohemagglutinin in transgenic tobacco

AU - Sturm, Arnd

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AU - Chrispeels, Maarten J.

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N2 - We used a heterologous system (transgenic Nicotiana tabacum L.) to investigate the processing, assembly and targeting of phytohemagglutinin (PHA), the lectin of the common bean, Phaseolus vulgaris L. In the bean, this glycoprotein accumulates in the protein bodies of the storage parenchyma cells in the cotyledons, and each polypeptide has a high-mannose glycan attached to Asn12 and a complex glycan on Asn60. The gene for PHA-L, dlec2, with 1200 basepairs (bp) 5′ upstream and 1600 bp 3′ downstream from the coding sequence was introduced into tobacco using Agrobacterium-mediated transformation (T. Voelker et al., 1987, EMBO J. 6, 3571-3577). Examination of thin sections of tobacco seeds by immunocytochemistry with antibodies against PHA showed that PHA-L accumulated in the amorphous matrix of the protein bodies in the embryo and endosperm. This localization was confirmed using a non-aqueous method to isolate the protein bodies from mature tobacco seeds. The biochemical analysis of tobacco PHA indicated that the signal peptide had been correctly removed, and that the polypeptides formed 6.4 S oligomers; tobacco PHA had a high-mannose glycan at Asn12 and a complex glycan at Asn60. The presence of the complex glycan shows that transport to the protein bodies was mediated by the Golgi complex. At seed maturity, a substantial portion of the PHA-L remained associated with the endoplasmic reticulum and the Golgi complex, as indicated by fractionation experiments using aqueous media and the presence of two high-mannose glycans on some of the polypeptides. Taken together, these data show that insertion of the nascent PHA into the endoplasmic reticulum, signal peptide processing, glycosylation, assembly into oligomers, glycan modification in the Golgi, and targeting of the protein occur faithfully in this heterologous system, although transport may not be as efficient as in bean cotyledons.

AB - We used a heterologous system (transgenic Nicotiana tabacum L.) to investigate the processing, assembly and targeting of phytohemagglutinin (PHA), the lectin of the common bean, Phaseolus vulgaris L. In the bean, this glycoprotein accumulates in the protein bodies of the storage parenchyma cells in the cotyledons, and each polypeptide has a high-mannose glycan attached to Asn12 and a complex glycan on Asn60. The gene for PHA-L, dlec2, with 1200 basepairs (bp) 5′ upstream and 1600 bp 3′ downstream from the coding sequence was introduced into tobacco using Agrobacterium-mediated transformation (T. Voelker et al., 1987, EMBO J. 6, 3571-3577). Examination of thin sections of tobacco seeds by immunocytochemistry with antibodies against PHA showed that PHA-L accumulated in the amorphous matrix of the protein bodies in the embryo and endosperm. This localization was confirmed using a non-aqueous method to isolate the protein bodies from mature tobacco seeds. The biochemical analysis of tobacco PHA indicated that the signal peptide had been correctly removed, and that the polypeptides formed 6.4 S oligomers; tobacco PHA had a high-mannose glycan at Asn12 and a complex glycan at Asn60. The presence of the complex glycan shows that transport to the protein bodies was mediated by the Golgi complex. At seed maturity, a substantial portion of the PHA-L remained associated with the endoplasmic reticulum and the Golgi complex, as indicated by fractionation experiments using aqueous media and the presence of two high-mannose glycans on some of the polypeptides. Taken together, these data show that insertion of the nascent PHA into the endoplasmic reticulum, signal peptide processing, glycosylation, assembly into oligomers, glycan modification in the Golgi, and targeting of the protein occur faithfully in this heterologous system, although transport may not be as efficient as in bean cotyledons.

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