title: Structural organization of distinct domains within the non-collagenous N-terminal region of collagen type XI.
abstract: Collagen XI is a heterotrimeric molecule found predominantly in heterotypic cartilage fibrils, where it is involved in the regulation of fibrillogenesis. This function is thought to involve the complex N-terminal domain. The goal of this current study was to examine its structural organization to further elucidate the regulatory mechanism. The amino-propeptide (alpha1-Npp) alone or with isoforms of the variable region were recombinantly expressed and purified by affinity and molecular sieve chromatography. Cys-1-Cys-4 and Cys-2-Cys-3 disulfide bonds were detected by liquid chromatography-tandem mass spectrometry. This pattern is identical to the homologous alpha2-Npp, indicating that the recombinant proteins were folded correctly. Anomalous elution on molecular sieve chromatography suggested that the variable region was extended, which was confirmed using rotary shadowing; the alpha1-Npp formed a globular "head" and the variable region an extended "tail." Circular dichroism spectra analysis determined that the alpha1-Npp comprised 33% beta-sheet, whereas the variable region largely comprised non-periodic structure. Taken together, these results imply that the alpha1-Npp cannot be accommodated within the core of the fibril and that the variable region and/or minor helix facilitates its exclusion to the fibril surface. This provides further support for regulation of fibril diameter by steric hindrance or by interactions with other matrix components that affect fibrillogenesis.
authors.affiliation: Shriners Hospitals for Children, Portland, Oregon 97201, USA.
authors.forename: K E
authors.initials: KE
authors.lastname: Gregory
authors.forename: J T
authors.initials: JT
authors.lastname: Oxford
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: J E
authors.initials: JE
authors.lastname: Gambee
authors.forename: S P
authors.initials: SP
authors.lastname: Gygi
authors.forename: R
authors.initials: R
authors.lastname: Aebersold
authors.forename: P J
authors.initials: PJ
authors.lastname: Neame
authors.forename: D E
authors.initials: DE
authors.lastname: Mechling
authors.forename: H P
authors.initials: HP
authors.lastname: Bächinger
authors.forename: N P
authors.initials: NP
authors.lastname: Morris
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003094:Collagen / Q000737:chemistry*; D004220:Disulfides; D006031:Glycosylation; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D003094:Collagen