Show simple item record

dc.contributor.authorKayser, Hartmut
dc.contributor.authorMann, Karlheinz
dc.contributor.authorMachaidze, Gia
dc.contributor.authorNimtz, Manfred
dc.contributor.authorRingler, Philippe
dc.contributor.authorMüller, Shirley A
dc.contributor.authorAebi, Ueli
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.
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
refterms.dateFOA2018-06-13T00:37:13Z
html.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.


Files in this item

Thumbnail
Name:
Publisher version
Thumbnail
Name:
Kayser et al_final.pdf
Size:
1.179Mb
Format:
PDF
Description:
original manuscript
Thumbnail
Name:
Kayser Table2.pdf
Size:
93.15Kb
Format:
PDF
Description:
Table 2

This item appears in the following Collection(s)

Show simple item record