Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family.

2.50
Hdl Handle:
http://hdl.handle.net/10033/86441
Title:
Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family.
Authors:
Kayser, Hartmut; Mann, Karlheinz; Machaidze, Gia; Nimtz, Manfred; Ringler, Philippe; Müller, Shirley A; Aebi, Ueli
Abstract:
The abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI approximately 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.
Affiliation:
Institut für Allgemeine Zoologie und Endokrinologie, Universität Ulm, Germany. hartmut.kayser@uni-ulm.de
Citation:
Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family. 2009, 389 (1):74-89 J. Mol. Biol.
Journal:
Journal of molecular biology
Issue Date:
29-May-2009
URI:
http://hdl.handle.net/10033/86441
DOI:
10.1016/j.jmb.2009.03.075
PubMed ID:
19361516
Type:
Article
Language:
en
ISSN:
1089-8638
Appears in Collections:
Publications from Division of Molekulare Struktur Biologie (MOSB)

Full metadata record

DC FieldValue Language
dc.contributor.authorKayser, Hartmuten
dc.contributor.authorMann, Karlheinzen
dc.contributor.authorMachaidze, Giaen
dc.contributor.authorNimtz, Manfreden
dc.contributor.authorRingler, Philippeen
dc.contributor.authorMüller, Shirley Aen
dc.contributor.authorAebi, Uelien
dc.date.accessioned2009-11-19T10:08:02Z-
dc.date.available2009-11-19T10:08:02Z-
dc.date.issued2009-05-29-
dc.identifier.citationIsolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family. 2009, 389 (1):74-89 J. Mol. Biol.en
dc.identifier.issn1089-8638-
dc.identifier.pmid19361516-
dc.identifier.doi10.1016/j.jmb.2009.03.075-
dc.identifier.urihttp://hdl.handle.net/10033/86441-
dc.description.abstractThe abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for arylphorins. The protein was further characterized as a weakly acidic (pI approximately 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.en
dc.language.isoenen
dc.subject.meshAmino Acid Sequenceen
dc.subject.meshAnimalsen
dc.subject.meshCarbohydrate Conformationen
dc.subject.meshChromatography, Gelen
dc.subject.meshElectrophoresis, Polyacrylamide Gelen
dc.subject.meshGlycosylationen
dc.subject.meshImaging, Three-Dimensionalen
dc.subject.meshInsect Proteinsen
dc.subject.meshLigandsen
dc.subject.meshModels, Molecularen
dc.subject.meshMolecular Sequence Dataen
dc.subject.meshMolecular Weighten
dc.subject.meshMothsen
dc.subject.meshPeptidesen
dc.subject.meshProtein Structure, Quaternaryen
dc.subject.meshSequence Homology, Amino Aciden
dc.subject.meshSpectrometry, Mass, Electrospray Ionizationen
dc.subject.meshSpectrometry, Mass, Matrix-Assisted Laser Desorption-Ionizationen
dc.subject.meshSpectrophotometry, Ultravioleten
dc.titleIsolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family.en
dc.typeArticleen
dc.contributor.departmentInstitut für Allgemeine Zoologie und Endokrinologie, Universität Ulm, Germany. hartmut.kayser@uni-ulm.deen
dc.identifier.journalJournal of molecular biologyen

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