Collagen XXVII Is Developmentally Regulated and Forms Thin Fibrillar Structures Distinct from Those of Classical Vertebrate Fibrillar Collagens

Darren A Plumb,Vivek Dhir,Aleksandr Mironov,Laila Ferrara,Richard Poulsom,Karl E Kadler,David J Thornton,Michael D Briggs,Raymond P Boot-Handford

doi:10.1074/jbc.c700021200

Abstract

We have generated an antiserum to the variable domain of mouse collagen XXVII, a recently discovered novel member of the fibrillar collagen family. Collagen XXVII protein was first detectable in the mouse at embryonic day 12.5 (E12.5). By E14.5, the protein localized to cartilage, developing dermis, cornea, the inner limiting membrane of the retina, and major arteries of the heart. However, at E18.5, collagen XXVII protein was no longer apparent in most tissues and appeared restricted mainly to cartilage where expression continued into adulthood. Type XXVII collagen immunolocalized to 10-nm-thick nonstriated fibrils that were distinct from fibrils formed by the classical fibrillar collagens. The transient nature of its expression and unusual fibrillar structure suggest that collagen XXVII plays a developmental role distinct from those of the classical fibrillar collagens.

Highlights

Fibrillar collagens are key structural components of the vertebrate skeleton and most other connective tissues
We have generated an antisera against the variable domain of mouse collagen XXVII and report that the protein was first detectable in mouse at embryonic day 12.5; that by E14.5,3 the protein localized to cartilage, developing dermis, cornea, inner limiting membrane of the eye, and aorta; that at E18.5, collagen XXVII protein was no longer apparent in most tissues and became restricted mainly to cartilage; that the expression continued into adulthood; and that type XXVII collagen immunolocalized to 10-nm-thick nonstriated fibrils distinct from fibrils formed by the classical fibrillar collagens
We have provided the first insight into the function of the collagen XXVII protein

Summary

EXPERIMENTAL PROCEDURES

Antisera—The variable domain of Col27A1 was amplified by PCR from mouse genomic DNA, sequenced and ligated in-frame into the pTrc-His bacterial expression vector (Invitrogen). Induced bacterial cells were pelleted, resuspended in 25 ml of ice-cold lysis buffer (1ϫ PBS, 1% (v/v) Triton X-100, and lysozyme to a final concentration of 0.25 mg/ml) containing a mixture of protease inhibitors (Roche Applied Science, catalog No 1836153, 1 tablet/50 ml of lysis buffer), and incubated on ice for 30 min. Nonspecific binding of primary or secondary antibodies was blocked by incubation in PBS containing 1% BSA (w/v) and 0.6% (v/v) goat serum (Dako A/S) for 1 h at room temperature. Each section was overlaid with primary antiserum in dilution buffer (PBS containing 1% BSA), and incubated at room temperature for 1 h. The secondary biotinylated goat anti-rabbit antiserum (Dako A/S) was applied at 1:400 in dilution buffer (PBS containing 1% BSA, 0.6% goat serum) for 1 h at room temperature. Images were 1), strong expression of collagen XXVII can be seen in the walls recorded on film

RESULTS

DISCUSSION