Denzinger, T; Przybylski, M; Savoca, R; Sonderegger, P (1997). Mass spectrometric characterisation of primary structure, sequence heterogeneity, and intramolecular disulfide loops of the cell adhesion protein axonin-1 from chicken. European Journal of Mass Spectrometry, 3(5):379-389.
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Axonin-1 is an axon-associated cell adhesion protein which exists as a membrane-attached form via a glycosylphosphatidyl-inositol anchor (GPI) and a secreted form. Axonin-1 promotes and regulates neurite outgrowth and plays a critical role as a growth cone sensor molecule in axonal pathways. The cDNA of axonin-1 from chicken codes for 1036 amino acids, including a hydrophobic N-terminal signal sequence and a C-terminal signal peptide. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of native chicken axonin-1 showed an apparent molecular weight of 130¿140 kDa. The characterisation of intact axonin-1 by matrix assisted laser desorption/ionisation mass spectrometry (MALDI-MS) yielded molecular masses of 122,750 and 125,100 Da which are consistent with the GPI-modified and an unmodified secreted form. The mass difference between the calculated molecular mass based on the amino acid sequence (107,640 Da) suggests modifications of up to 15 kDa whichare assumed to be mainly due to glycosylation. In this study, the detailed structural characterisation of axonin-1 in its secreted GPI-anchorless form was carried out. Mass spectrometric peptide mapping analyses using the endoproteinases Lys¿C and Asp¿N revealed the complete amino acid sequence, the N- and C-terminal heterogeneities and, in conjunction with reductive cleavage, the intramolecular disulphide-loop pattern. Furthermore, the preliminary results on the location of N-glycosylation sites could be obtained. In addition, proteolytic fragments were isolated by high performance liquid chromatography (HPLC), identified by MALDI-MS and further digested with trypsin. These results revealed the presence of a blocked N- terminus (pyroglutamyl pQ22) and provided the processed C-terminus as M1004 for the native, GPI-unmodified form of axonin-1.
|Item Type:||Journal Article, refereed, original work|
|Communities & Collections:||04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
|Dewey Decimal Classification:||570 Life sciences; biology|
|Deposited On:||11 Feb 2008 12:20|
|Last Modified:||05 Apr 2016 12:17|
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