Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Dec 1;350(4):1032-7.
doi: 10.1016/j.bbrc.2006.09.147. Epub 2006 Oct 5.

The NC16A Domain of Collagen XVII Plays a Role in Triple Helix Assembly and Stability

Affiliations
Free PMC article

The NC16A Domain of Collagen XVII Plays a Role in Triple Helix Assembly and Stability

Françoise Van den Bergh et al. Biochem Biophys Res Commun. .
Free PMC article

Abstract

Collagen XVII/BP180 is a transmembrane constituent of the epidermal anchoring complex. To study the role of its non-collagenous linker domain, NC16A, in protein assembly and stability, we analyzed the following recombinant proteins: the collagen XVII extracellular domain with or without NC16A, and a pair of truncated proteins comprising the COL15-NC15 stretch expressed with or without NC16A. All four proteins were found to exist as stable collagen triple helices; however, the two missing NC16A exhibited melting temperatures significantly lower than their NC16A-containing counterparts. Protein refolding experiments revealed that the rate of triple helix assembly of the collagen model peptide GPP(10) is greatly increased by the addition of an upstream NC16A domain. In summary, the NC16A linker domain of collagen XVII exhibits a positive effect on both the rate of assembly and the stability of the adjoining collagen structure.

Figures

Fig. 1
Fig. 1. Schematic representation of recombinant proteins of collagen XVII
The extracellular domain of type XVII collagen (top diagram) consists of 15 collagenous domains (solid boxes) and 16 non-collagenous domains (horizontal lines). Also shown are schematic representations of the recombinant proteins used in this study. The corresponding AA positions are indicated for the termini of each protein (from GenBank accession NM_130778). The lengths (in AA) of sec180, sec180-ΔN16, sec180-trunc and trunc-ΔN16 are 1008, 931, 331 and 254, respectively. The His-tag, when present, adds 13 AA to the length of the protein. TM, transmembrane domain; pos, position.
Fig. 2
Fig. 2. Cross-link analysis of collagen XVII recombinant proteins
The collagen XVII truncation mutants were subjected to chemical cross-linking using disuccinimidyl suberate (DSS). Panel A, This figure is a Western blot of sec180 and sec180-ΔN16 treated with no DSS (lanes 1 and 4) or with DSS at 0.1 mM (lanes 2 and 5) or 0.5 mM (lanes 3 and 6). Panel B, This is a Western blot of sec180-trunc and trunc-ΔN16-cHis at low and high concentrations treated with no DSS (lanes 1, 4 and 7) or with DSS at 0.1 mM (lanes 2, 5 and 8) or 0.5 mM (lanes 3, 6 and 9). In both panels A & B, the positions of the monomer, dimer and trimer forms are indicated by "M", "D", and "T", respectively. The positions of the molecular weight markers are shown to the left of the blots.
Fig. 3
Fig. 3. Thermal denaturation analysis of collagen XVII recombinant proteins
Panel A, This panel shows representative thermal denaturation curves that were generated by monitoring trypsin resistance. Panel B. The bar graph displays the mean melting temperature (Tm; 4–6 experiments) and standard deviation obtained for each protein. Panel C. Each recombinant protein was incubated with or without trypsin at 4 °C. Shown in the bar graph are the mean percentages of protein resistant to trypsin (4–8 digestions) and the corresponding standard deviations.
Fig. 4
Fig. 4. Refolding of model proteins
Panel A, This plot shows refolding curves for the model protein NC16A-GPP10 (closed circles) and the synthetic collagen peptide sGPP10 (closed triangles). Panel B, This plot shows the logarithm of initial rate (in M/sec) of triple helix formation of sGPP10 as a function of the logarithm of total chain concentration (in M).

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

Publication types

LinkOut - more resources

Feedback