Synthesis and Folding of Native Collagen III Model Peptides

Henkel, Werner; Vogl, Thomas; Echner, Hartmut; Voelter, Wolfgang; Urbanke, Claus; Schleuder, Detlef; Rauterberg, Jürgen

doi:10.1021/bi9905157

Cited by 40 publications

(36 citation statements)

References 38 publications

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“…To test this hypothesis, the thermal stabilities and lengths of (1) n and (2) n were measured in aqueous methanol, where collagen triple helices are highly stable (39). Indeed, increases in both thermal stability (Fig.…”

Section: Resultsmentioning

confidence: 99%

Self-assembly of synthetic collagen triple helices

Kotch

Raines

2006

Proc. Natl. Acad. Sci. U.S.A.

280

232

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Collagen is the most abundant protein in animals and the major component of connective tissues. Although collagen isolated from natural sources has long served as the basis for some biomaterials, natural collagen is difficult to modify and can engender pathogenic and immunological side effects. Collagen comprises a helix of three strands. Triple helices derived from synthetic peptides are much shorter (<10 nm) than natural collagen (Ϸ300 nm), limiting their utility. Here, we describe the synthesis of short collagen fragments in which the three strands are held in a staggered array by disulfide bonds. Data from CD spectroscopy, dynamic light scattering, analytical ultracentrifugation, atomic force microscopy, and transmission electron microscopy indicate that these ''sticky-ended'' fragments self-assemble via intermolecular triple-helix formation. The resulting fibrils resemble natural collagen, and some are longer (>400 nm) than any known collagen. We anticipate that our self-assembly strategy can provide synthetic collagen-mimetic materials for a variety of applications.biomaterial ͉ coiled-coil ͉ nanotechnology ͉ cystine knot ͉ peptide C ollagen constitutes one-third of the human proteome, including three-quarters of the dry weight of human skin. Its high natural abundance and intrinsic plasticity have spurred the development of collagen as a biomaterial (1, 2). The most common source of clinical collagen is now Bos taurus, the domestic cow. Unfortunately, bovine collagen can illicit deleterious pathological and immunological effects when transplanted into humans (3-5). Moreover, the preparation of enriched solutions of natural collagen is problematic (6), and its sitespecific covalent modification is not feasible. We suspected that synthetic chemistry could offer a solution to these problems.The quaternary structure of collagen comprises three strands that wrap around one another to form a triple helix (7). Each strand has the repeating sequence XaaYaaGly, with the most abundant triplet being ProHypGly [Hyp ϭ (2 S,4R)-4-hydroxyproline] (8). The folding of (XaaYaaGly) nՅ10 peptides into blunt-ended triple helices has been investigated thoroughly (7). Although such triple helices are biomaterial candidates (9-12), they are limited to the length of a synthetic peptide (Ͻ10 nm), which is much shorter than natural collagen (Ϸ300 nm). The polymerization of (XaaYaaGly) 10 peptides has afforded long strands that adopt triple-helical structure but have high polydispersity (13).Molecular self-assembly underlies the ''bottom-up'' approach to macromolecular design, wherein a desirable structure forms spontaneously through noncovalent interactions (14, 15). Selfassembling peptides and proteins have been designed to serve as materials for biological and nanotechnological applications (16,17). Using the self-assembly approach, Woolfson and coworkers (18,19) have produced fibers with a design based on a dimeric coiled-coil structure. Likewise, fibrous peptides based on the natural protein elastin (20) and de novo building b...

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Section: Resultsmentioning

confidence: 99%

Self-assembly of synthetic collagen triple helices

Kotch

Raines

2006

Proc. Natl. Acad. Sci. U.S.A.

280

232

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“…Alternatively, C 16 -fTHP-10 may aggregate in an unusual fashion, destabilizing the triple helix. Such effects may be explored elsewhere using differential scanning calorimetry (55)(56)(57)(58). Because of its anomalous behavior, C 16 -fTHP-10 was omitted from further study.…”

Section: Methodsmentioning

confidence: 99%

The Roles of Substrate Thermal Stability and P2 and P1′ Subsite Identity on Matrix Metalloproteinase Triple-helical Peptidase Activity and Collagen Specificity

Minond

Lauer‐Fields

Čudić

et al. 2006

Journal of Biological Chemistry

134

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The hydrolysis of collagen (collagenolysis) is one of the committed steps in extracellular matrix turnover. Within the matrix metalloproteinase (MMP) family distinct preferences for collagen types are seen. The substrate determinants that may guide these specificities are unknown. In this study, we have utilized 12 triple-helical substrates in combination with 10 MMPs to better define the contributions of substrate sequence and thermal stability toward triple helicase activity and collagen specificity. In general, MMP-13 was found to be distinct from MMP-8 and MT1-MMP(⌬279 -523), in that enhanced substrate thermal stability has only a modest effect on activity, regardless of sequence. This result correlates to the unique collagen specificity of MMP-13 compared with MMP-8 and MT1-MMP, in that MMP-13 hydrolyzes type II collagen efficiently, whereas MMP-8 and MT1-MMP are similar in their preference for type I collagen. In turn, MMP-1 was the least efficient of the collagenolytic MMPs at processing increasingly thermal stable triple helices and thus favors type III collagen, which has a relatively flexible cleavage site. Gelatinases (MMP-2 and MMP-9(⌬444 -707)) appear incapable of processing more stable helices and are thus mechanistically distinct from collagenolytic MMPs. The collagen specificity of MMPs appears to be based on a combination of substrate sequence and thermal stability. Analysis of the hydrolysis of triple-helical peptides by an MMP mutant indicated that Tyr 210 functions in triple helix binding and hydrolysis, but not in processing triple helices of increasing thermal stabilities. Further exploration of MMP active sites and exosites, in combination with substrate conformation, may prove valuable for additional dissection of collagenolysis and yield information useful in the design of more selective MMP inhibitors.Current studies identify at least 25 different collagen types, each with a specific role in the extracellular matrix (1, 2). The hydrolysis of collagen (collagenolysis) is one of the committed steps in extracellular matrix turnover (3). The triple-helical structure of collagen renders it resistant to most proteases. In vertebrates, enzymes capable of cleaving the triple-helical structure include cathepsin K and collagenolytic matrix metalloproteinase (MMP) 2 family members. One or more of the interstitial collagens (types I-III) are hydrolyzed within their triple-helical domain by MMP-1, MMP-2, MMP-8, MMP-13, MMP-18, MT1-MMP (MMP-14), and MT2-MMP (MMP-15) (4, 5). MMP-9 cleaves the triple helix of types V and XI collagen (6) but not of types I-III (7).Types I-III collagen are all fibrillar interstitial collagens, but differences in their sequences, glycosylation patterns, and tissue distribution have long been documented (1, 8 -10). For example, type I collagen has a low level of glycosylation and is found in skin, bone, cornea, and tendon, whereas type II collagen has much higher levels of glycosylation and is found in cartilage (1, 9). In a similar fashion to type I collagen, type III col...

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“…Edman degradation sequence analysis was performed on an Applied Biosystems 477A protein sequencer/120A analyzer as described (31) for "embedded" (non-covalent) sequencing. MALDI-MS was performed on a Hewlett-Packard G2025A mass spectrometer using either a sinapinic acid or 2,5-dihydroxybenzoic acid/2-hydroxy-5-methoxy-benzoic acid (9:1, v/v) matrix (32 Circular Dichroism Spectroscopy-Circular dichroism spectra were recorded over the range ϭ 190 -250 nm on a JASCO J-600 using a 10-mm path length quartz cell. Thermal transition curves were obtained by recording the molar ellipticity ([⌰]) at ϭ 225 nm while the temperature was continuously increased in the range of 5-80°C at a rate of 0.2°C/min.…”

Section: Methodsmentioning

confidence: 99%

Selective Hydrolysis of Triple-helical Substrates by Matrix Metalloproteinase-2 and -9

Lauer‐Fields

Sritharan

Stack

et al. 2003

Journal of Biological Chemistry

114

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MMP-1, -3, -13, or -14. ␣1(V)436 -447 fTHP and a general fluorogenic THP were used to screen for triple-helical peptidase activity in ␣ 2 ␤ 1 integrin-stimulated melanoma cells. Binding of the ␣ 2 ␤ 1 integrin resulted in the production of substantial triple-helical peptidase activity, the majority (>95%) of which was non-MMP-2/-9. THPs were found to provide highly selective substrates for members of the MMP family and can be used to evaluate active MMP production in cellular systems.The role of proteases, particularly metalloproteases, in the tumor cell invasion process is multifaceted. Members of the matrix metalloproteinase (MMP) 1 family have been implicated in primary and metastatic tumor growth, angiogenesis, and degradation of ECM components during tumor cell invasion (1-4). MMP expression is widespread in common human tumors, with MMP

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Synthesis and Folding of Native Collagen III Model Peptides

Cited by 40 publications

References 38 publications

Self-assembly of synthetic collagen triple helices

Self-assembly of synthetic collagen triple helices

The Roles of Substrate Thermal Stability and P2 and P1′ Subsite Identity on Matrix Metalloproteinase Triple-helical Peptidase Activity and Collagen Specificity

Selective Hydrolysis of Triple-helical Substrates by Matrix Metalloproteinase-2 and -9

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