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.2012 Mar 27;51(12):2417-24.
doi: 10.1021/bi2019139. Epub 2012 Mar 15.

Peptidyl 3-hydroxyproline binding properties of type I collagen suggest a function in fibril supramolecular assembly

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Peptidyl 3-hydroxyproline binding properties of type I collagen suggest a function in fibril supramolecular assembly

David M Hudson et al. Biochemistry..

Abstract

Proline residues in collagens are extensively hydroxylated post-translationally. A rare form of this modification, (3S,2S)-l-hydroxyproline (3Hyp), remains without a clear function. Disruption of the enzyme complex responsible for prolyl 3-hydroxylation results in severe forms of recessive osteogenesis imperfecta (OI). These OI types exhibit a loss of or reduction in the level of 3-hydroxylation at two proline residues, α1(I) Pro986 and α2(I) Pro707. Whether the resulting brittle bone phenotype is caused by the lack of the 3-hydroxyl addition or by another function of the enzyme complex is unknown. We have speculated that the most efficient mechanism for explaining the chemistry of collagen intermolecular cross-linking is for pairs of collagen molecules in register to be the subunit that assembles into fibrils. In this concept, the exposed hydroxyls from 3Hyp are positioned within mutually interactive binding motifs on adjacent collagen molecules that contribute through hydrogen bonding to the process of fibril supramolecular assembly. Here we report observations on the physical binding properties of 3Hyp in collagen chains from experiments designed to explore the potential for interaction using synthetic collagen-like peptides containing 3Hyp. Evidence of self-association was observed between a synthetic peptide containing 3Hyp and the CB6 domain of the α1(I) chain, which contains the single fully 3-hydroxylated proline. Using collagen from a case of severe recessive OI with a CRTAP defect, in which Pro986 was minimally 3-hydroxylated, such binding was not observed. Further study of the role of 3Hyp in supramolecular assembly is warranted for understanding the evolution of tissue-specific variations in collagen fibril organization.

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Figures

Figure 1
Figure 1. The 3Hyp probe recognizes and binds type I collagen from multiple tissue sources
Intact type I collagen chains were separated on 6% SDS-PAGE and either stained with Coomassie blue (stained gel) or transblotted to nitrocellulose for far Western analysis. Blots were probed using 3Hyp probe (3Hyp), Pro probe (Pro), or extravidin-HRP without prior incubation with a primary probe (HRP). Lanes 1, human bone collagen; lanes 2, chicken skin collagen; lanes 3, chicken tendon collagen.
Figure 2
Figure 2. Interaction with CNBr-digested collagen peptides is specific for the 3Hyp probe and is enhanced by the 3-hydroxyl modification
Peptide map of CNBr-digested type I collagen chains (A). Replicate sample lanes of CNBr-digested human bone matrix were separated on 12% SDS-PAGE (B) and stained with Coomassie blue (stained gel), or transblotted to nitrocellulose and probed individually using the following synthetic peptides: extravidin-HRP without prior incubation with a primary probe (HRP), CB8 probe (CB8), A2 probe (A2), Pro probe (Pro), or 3Hyp probe (3Hyp). The predominant CNBr-digested collagen peptides are indicated on the far left. The predicted CB6-containing cross-linked peptides are indicated on the far right (cross-links are depicted as ×), see text for details. The N-terminal telopeptides of α1(I) and α2(I) are indicated as α1(I)CB1 and α2(I)CB1, respectively. Replicate sample lanes of CNBr-digested human bone matrix were analyzed with Pro probe (Pro) or 3Hyp probe (3Hyp) in the presence of 2 M glucose (Glc) to inhibit hydrogen bond formation (C). The protein standard sizes are shown in kDa.
Figure 3
Figure 3. Binding epitope for 3Hyp probe is located within the CB6 region of collagen α1(I)
Full scan spectra from LC-MS profile (A) and MS/MS fragmentation profile (B) of in-gel trypsin digest of a representative reactive band from far Western analysis (Figure 2B) reveals the presence of the 3Hyp-containing CB6 peptide at multiple sites on the 12% SDS-PAGE. Far Western blots of isolated CB6 (lanes 1) and CB3 (lanes 2) from α1(I) collagen of human bone were probed using 3Hyp probe (3Hyp), Pro probe (Pro), or extravidin-HRP without prior incubation with a primary probe (HRP) (C).
Figure 4
Figure 4. Self-association is not observed from OI bone in which Pro986 is not 3-hydroxylated
Full scan spectra from LC-MS profile (A) and MS/MS fragmentation profile (B) of in-gel trypsin digests confirm that Pro986 is not hydroxylated in the OI bone. Far Western blots of CNBr-digested fetal control (lanes 1) and OI (lanes 2) bone were probed using 0.2 μg/mL 3Hyp probe (3Hyp ), 0.5 μg/mL Pro probe (Pro), or extravidin-HRP without incubation with a primary probe (HRP) (C). These data are consistent with our hypothesis that intermolecular recognition resulting in self-association is specific to collagen peptides containing 3Hyp at position 986.
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