Conformational study of a collagen peptide by 1H NMR spectroscopy: observation of the 14N-1H spin-spin coupling of the Arg guanidinium moiety in the triple-helix structure

AbstractCB2, a CNBr peptide of 36 residues from type I collagen α1(I) chain has been studied by NMR spectroscopy as a function of temperature. At low temperature, the guanidinium protons of Arg9 showed sharp 1:1:1 NMR triplets around 6.95 ppm, characteristic of 14N coupled protons (1JNH=52 Hz) when the quadrupolar relaxation rate is drastically reduced. These spectral characteristics and the low temperature coefficient of the 1:1:1 triplets (Δδ/ΔT of −3.6 ppb/°C) suggest that the H atoms of the protonated guanidinium moiety of Arg9 in the triple helix are slowly exchanging with bulk water, most likely involved in hydrogen bonds. On the basis of conformational energy computations on a model segment of type I collagen (Vitagliano, L., Némethy, G., Zagari, A. and Scheraga, H.A. (1993) Biochemistry 32, 7354–7359), similar to CB2, our data could indicate that the guanidinium group of Arg9 form hydrogen bonds with a backbone carbonyl of an adjacent chain probably by using the Nϵ hydrogen, leaving the four Nη hydrogens bound to water molecules that must be in slow exchange with bulk water and that could therefore be considered structural elements of the trimeric α1(I) CB2 triple helix. The behaviour of Arg9 has been investigated also in terms of equilibrium between random monomer and helical trimer conformations controlled by temperature. The thermal unfolding process was found to be reversible and the melting point resulted to be 17°C

N-acetylcysteine treatment ameliorates the skeletal phenotype of a mouse model of diastrophic dysplasia

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by mutations in SLC26A2, a cell membrane sulfate-chloride antiporter. Sulfate uptake impairment results in low cytosolic sulfate, leading to cartilage proteoglycan (PG) undersulfation. In this work, we used the dtd mouse model to study the role of N-acetyl-l-cysteine (NAC), a well-known drug with antioxidant properties, as an intracellular sulfate source for macromolecular sulfation. Because of the important pre-natal phase of skeletal development and growth, we administered 30 g/l NAC in the drinking water to pregnant mice to explore a possible transplacental effect on the fetuses. When cartilage PG sulfation was evaluated by high-performance liquid chromatography disaccharide analysis in dtd newborn mice, a marked increase in PG sulfation was observed in newborns from NAC-treated pregnancies when compared with the placebo group. Morphometric studies of the femur, tibia and ilium after skeletal staining with alcian blue and alizarin red indicated a partial rescue of abnormal bone morphology in dtd newborns from treated females, compared with pups from untreated females. The beneficial effect of increased macromolecular sulfation was confirmed by chondrocyte proliferation studies in cryosections of the tibial epiphysis by proliferating cell nuclear antigen immunohistochemistry: the percentage of proliferating cells, significantly reduced in the placebo group, reached normal values in dtd newborns from NAC-treated females. In conclusion, NAC is a useful source of sulfate for macromolecular sulfation in vivo when extracellular sulfate supply is reduced, confirming the potential of therapeutic approaches with thiol compounds to improve skeletal deformity and short stature in human DTD and related disorder

XXV Meeting THE ITALIAN SOCIETY FOR THE STUDY OF CONNECTIVE TISSUES (SISC)

XXV Meeting THE ITALIAN SOCIETY FOR THE STUDY OF CONNECTIVE TISSUES (SISC

Mapping Hsp47-binding site(s) using CNBr peptides derived from type I and II collagen

As a crucial molecular chaperone in Collagen biosynthesis, Hsp47 interacts with the nascent form as well as the mature triple-helical form of procollagen. The location(s) of Hsp47 binding sites on the collagen molecule are, as yet, unknown. We have examined the substrate specificity of Hsp47 in vitro using well-characterized CNBr peptide fragments of type I and type II Collagen along with radiolabeled, recombinant Hsp47. Interaction of these peptides with Hsp47 bound to collagen-coated microtiter wells showed several binding sites for Hsp47 along the length of the alpha1 and alpha2 chains of type I Collagen and the alpha1 chain of type II collagen, with the N-terminal regions showing the strongest affinities. The latter observation was also supported by the results of a ligand-blot assay. Except for two peptides in the alpha2(I) chain, peptides that showed substantial binding to Hsp47 did so in their triple-helical and not random-coil form. Unlike earlier studies that used peptide models for Collagen, the results obtained here on fragments of type I and type II collagen identify, for the first time, binding of Hsp47 to specific regions of the collagen molecule. They also point to additional structural requirements for Hsp47 binding besides the known preference for third-position Arg residues and the triple-helical conformation

Possible role of overglycosylation in the type I collagen triple helical domain in the molecular pathogenesis of osteogenesis imperfecta.

The underlying defect in patients affected by a form of osteogenesis imperfecta (OI) clarified at the molecular level regards the amount or the structure of type I collagen synthesized. This leads to a decreased and/or abnormal mineral deposition in bone and affects bone mass and/or strength. Abnormal interactions between collagen molecules in the presence of mutant trimers could give rise to abnormal fibrils, which, in turn, can determine incorrect interactions with noncollagenous matrix macromolecules. The interactions can be disturbed or modulated by an abnormal distribution on the collagen fibril surface of electrically charged or hydrophobic groups, or by an increased presence of sugar moieties linked to hydroxylysyl residues due to chain post-translational overmodifications (lysyl overhydroxylation and hydroxylysyl overglycosylation) of the portion of the triple helical domain of abnormal type I collagen molecules N-terminal with respect to the defect localization

The Structural characterization and bilirubin-binding properties of albumin Herborn, a [Lys240->Glu] albumin mutant

We report the molecular defect of albumin Herborn, a new genetic variant of human serum albumin which has been found in Germany. Isoelectric focusing analysis of CNBr fragments from the purified variant allowed us to localize the mutation in fragment CNBr 3 (residues 124–298). This fragment was isolated on a preparative scale and subjected to tryptic and V8 protease digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution Lys240→Glu. The -2 charge change of albumin Herborn, which is probably due to a A→G transition in the first position of the corresponding codon in the structural gene, has no significant effect on its electrophoretic mobility under non-denaturating conditions. Therefore we have assumed that residue 240, which has been implicated in the bilirubin primary binding site (Jacobsen, C. (1978) Biochem. J. 171, 453–459), is buried. The binding of bilirubin and biliverdin by albumin Herborn was quantified using the fluorescence quenching method. The apparent equilibrium association constants (Ka±SD) and the number of high-affinity binding sites (n) of the defatted variant for bilirubin and biliverdin were Ka= 1.03 ± 0.18 × 108 M−1, n= 1.07; and Ka= 7.48 ± 1.10 × 106 M−1, n= 1.01, respectively. The Ka values are about 93.3% and 99.1% of the values found for the normal protein under the same conditions. These results strongly suggest that Lys240 of human serum albumin is not the basic residue involved in ion pairing with one of the carboxylate groups of bilirubin at its high-affinity site

Collagen fibril structure is affected by collagen concentration and decorin

Abstract Collagen fibrils were obtained in vitro by aggregation from acid-soluble type I collagen at different initial concentrations and with the addition of decorin core or intact decorin. All specimens were observed by scanning electron microscopy and atomic force microscopy. In line with the findings of other authors, lacking decorin, collagen fibrils undergo an extensive lateral association leading to the formation of a continuous three-dimensional network. The addition of intact decorin or decorin core was equally effective in preventing lateral fusion and restoring the normal fibril appearance. In addition, the fibril diameter was clearly dependent on the initial collagen concentration but not on the presence/absence of proteoglycans. An unusual fibril structure was observed as a result of a very low initial collagen concentration, leading to the formation of huge, irregular superfibrils apparently formed by the lateral coalescence of lesser fibrils, and with a distinctive coil-structured surface. Spots of incomplete fibrillogenesis were occasionally found, where all fibrils appeared made of individual, interwined subfibrils, confirming the presence of a hierarchical association mechanism