Solid-phase synthesis of triple-helical collagen-model peptides

Fields, Cynthia G.; Grab, B.; Lauer, J.; Miles, Andrew; Yu, Ying‐Ching; Fields, Gregg B.

doi:10.1007/bf00131080

Cited by 26 publications

(23 citation statements)

References 45 publications

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“…All THPs were purified by a two-step reversed-phase HPLC procedure (15). The branch portion of the THPs was characterized by mass spectrometry, whereas the collagen-like sequence was characterized by Edman degradation sequence analysis (13,16). THPs were homogeneous by analytical reversed-phase HPLC.…”

Section: Methodsmentioning

confidence: 99%

Defining the domains of type I collagen involved in heparin- binding and endothelial tube formation

Sweeney

Guy

Fields

et al. 1998

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

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105

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Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiquitous cell adhesion mechanism. However, the HSPG binding sites on type I collagen are unknown. Previously we mapped heparin binding to the vicinity of the type I collagen N terminus by electron microscopy. The present study has identified type I collagen sequences used for heparin binding and endothelial cell-collagen interactions. Using affinity coelectrophoresis, we found heparin to bind as follows: to type I collagen with high affinity ( Cell surface proteoglycan interactions with type I collagen are likely a ubiquitous mechanism of cell adhesion, yet the interactive sites on these molecules are undefined. Consideration of the complexity of type I collagen structure is relevant to understanding its interactions with heparan sulfate proteoglycans (HSPGs) (see refs. 1 and 2 for review). Type I collagen is secreted as procollagen, which, after proteolytic cleavages, yields the triple-helical monomer composed of two ␣1 and one ␣2 chains. These monomers assemble in a regular staggered fashion into fibrils, which display the repeating D-period pattern, consisting of fine crossfibril bands (called the a, b, c, d, and e bands) in positively stained electron microscopy preparations.Triple-helical type I collagen conformation is necessary for its high-affinity binding to HSPGs, or to heparin, a chemical analog of its heparan sulfate chains (3-5). Furthermore, the C-terminal triple-helical fragment of type I collagen, generated by vertebrate collagenase treatment, showed a higher affinity for heparin than the did the N-terminal fragment (4).To localize more precisely the heparin-binding regions on type I collagen, we studied complexes between collagen monomers and heparin-albumin-gold particles by electron microscopy (6), and we observed heparin binding primarily to a region on the triple helix near the procollagen N terminus. In collagen fibrils, heparin-gold bound to the a bands region within each D-period, which is consistent with an N-terminal heparinbinding site on its monomers. The resolution of the mapping technique was insufficient to assign heparin-binding function to any particular protein sequence. Thus, amino acid sequences were inspected in relation to the heparin-binding locations observed by electron microscopy, searching for basic domains that might be suitable as heparin-binding sites. A highly basic sequence was found near the procollagen N terminus, and within the a bands fibril region, corresponding to amino acid residues 87-92 of the rat ␣1 chain. To test the heparin-binding function of this or other sites of type I collagen, here we have studied how mimetic triple-helical peptides (THPs) including various collagen sequences interact with heparin. We have also explored the function of these sequences in endothelial cell interactions with type I collagen during endothelial tube formation.

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Section: Methodsmentioning

confidence: 99%

Defining the domains of type I collagen involved in heparin- binding and endothelial tube formation

Sweeney

Guy

Fields

et al. 1998

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

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105

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“…Amino acids are of the L configuration (except for Gly). THPs were synthesized and purified by methods described previously in our laboratory (28,29,48). The synthesis and characterization of the ␣1(I)772-786 THP and ␣1(I)772-786 single-stranded peptide have been published (31).…”

Section: Methodsmentioning

confidence: 99%

“…Several methods have been described by which THPs incorporating collagen-like sequences and with T m values greater than 30°C can be synthesized. Our laboratory has developed a solid phase THP synthetic method that features a COOH-terminal Lys covalent branch (27)(28)(29). A variety of THPs have been constructed for the study of cellular recognition of collagen (27, 30 -32), including one that incorporates the collagenase cleavage site in type I collagen (31).…”

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confidence: 99%

Hydrolysis of Triple-helical Collagen Peptide Models by Matrix Metalloproteinases

Lauer‐Fields

Tuzinski

Shimokawa

et al. 2000

Journal of Biological Chemistry

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120

138

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The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis. To study the mechanisms of MMP action on collagenous substrates, we have constructed homotrimeric triplehelical peptide (THP) models of the collagenase cleavage sites in types I and II collagen. The THPs incorporate either the ␣1(I)772-786 or the ␣1(II)772-783 sequence. The ␣1(I)772-786 and ␣1(II)772-783 THPs were hydrolyzed by MMP-1 at the Gly-Ile and Gly-Leu bonds, respectively, analogous to the bonds cleaved in corresponding native collagens. Thus, the THPs contained all necessary information to direct MMP-1 binding and proteolysis. Subsequent investigations using the ␣1 ( . We propose that the COOH-terminal domain of MMPs is necessary for orienting whole, native collagen molecules but may not be necessary for binding to and cleaving a THP. This proposal is consistent with the large distance between the MMP-1 catalytic and COOHterminal domains observed by three-dimensional structural analysis and supports previous suggestions that the features of the catalytic domain contribute significantly toward enzyme specificity. The matrix metalloproteinase (MMP)1 family plays an integral role in both normal and pathological connective tissue remodeling (1). This accelerated local turnover of the extracellular matrix can be found in such diverse diseases as arthritis, glomerulonephritis, periodontal disease, and tumor cell invasion and metastasis (2, 3). There are suggestions that, based on their substrate specificities, MMPs may act on the matrix components in a sequential manner (4). In particular, MMPs are believed to initiate interstitial collagen catabolism and participate in denatured collagen (gelatin) degradation. Interstitial collagens (types I-III) are hydrolyzed at a single locus by MMP-1 (collagenase 1) (5), MMP-8 (collagenase 2) (5), and MMP-18 (collagenase 4) (6). MMP-2 hydrolyzes type I collagen with a similar k cat and a slightly higher K m values than those of MMP-1 at the same single locus (7). MMP-3 binds to type I collagen but does not cleave the triple-helical domain (8, 9). MMP-13 (collagenase 3) hydrolyzes type II collagen at both the same site as MMP-1 and at a site three residues proximal (the 778 -779 bond) (10, 11). MMP-13 cleaves types I and III collagen as well, but not as rapidly as type II (12). Membrane type 1 MMP (MMP-14) also cleaves interstitial collagens (13). MMP-14 is 5-7-fold less efficient at hydrolyzing type I collagen than MMP-1 (13). Several investigations have attempted to define which MMP domains are required for collagenolytic activity. Deletion of the COOH-terminal hemopexin-like domain from MMP-1 (residues 243-450), MMP-8 (residues 243-467), or MMP-13 (residues 249 -451) results in a loss of collagenolytic activity (9,(13)(14)(15)(16). A chimeric MMP-8 whose linker region (16 residues) between the catalytic and COOH-terminal domains is replaced with the corresponding MMP-3 sequence (25 residues) lost activity t...

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“…The Lys-Lys template approach has extensively been exploited in the solid-phase synthesis of homotrimeric collagenous peptides [14] and even of a heterotrimer [15], despite the serious difficulties encountered in the synthesis and purification of such high molecular weight polypeptides. Since both these templates are highly flexible structures, the use of a conformationally constrained molecule, i.e.…”

Section: Introductionmentioning

confidence: 99%

Design and synthesis of heterotrimeric collagen peptides with a built‐in cystine‐knot Models for collagen catabolism by matrix‐metalloproteases

et al. 1996

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A heterotrimeric collagen peptide was designed and synthesized which contains the collagenase cleavage site (P4-P'+/t0) of type I collagen linked to a C-terminal cystine-knot, and N-terminally extended with (Gly-Pro-Hyp)s triplets for stabilization of the triple-helical conformation. By employing a newly developed regioselective cysteine pairing strategy based exclusively on thiol disulfide exchange reactions, we succeeded in assembling in high yields and in a reproducible manner the triplestranded cystine peptide. While the single chains showed no teodeney to self-association into triple helices, the heterotrimer (cxltz2~xl') was found to exhibit a typical collagen-like CD spectrum at room temperature and a melting temperature (Tin) of

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Solid-phase synthesis of triple-helical collagen-model peptides

Cited by 26 publications

References 45 publications

Defining the domains of type I collagen involved in heparin- binding and endothelial tube formation

Defining the domains of type I collagen involved in heparin- binding and endothelial tube formation

Hydrolysis of Triple-helical Collagen Peptide Models by Matrix Metalloproteinases

Design and synthesis of heterotrimeric collagen peptides with a built‐in cystine‐knot Models for collagen catabolism by matrix‐metalloproteases

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