Synthesis of Poly-(<scp>l</scp>-prolyl-<scp>l</scp>-prolylglycyl) of Defined Molecular Weights

Sakakibara, Shumpei; Kishida, Yukichi; Kikuchi, Yasuo; Sakai, Ryutaro; Kakiuchi, Kinji

doi:10.1246/bcsj.41.1273

Cited by 149 publications

(116 citation statements)

References 6 publications

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“…To elucidate the structural requirements of collagen for interaction with HSP47, we first performed a solid-phase pull down assay using immobilized commercially available peptides that could mimic the collagen-like structure. Poly-L-proline, polyglycine, (ProPro-Gly) 5 , and (Pro-Pro-Gly) 10 (17,18) were used for the initial assay. The E. coli-harboring expression plasmid encoding rHSP47 was cultured, and the protein expression was induced moderately so as not to accumulate too much rHSP47 as compared with other endogenous proteins.…”

Section: Resultsmentioning

confidence: 99%

Substrate Recognition of Collagen-specific Molecular Chaperone HSP47

Koide¹,

Asada²,

Nagata³

1999

Journal of Biological Chemistry

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Prior to secretion, procollagen molecules are correctly folded to triple helices in the endoplasmic reticulum (ER). HSP47 specifically associates with procollagen in the ER during its folding and/or modification processes and is thought to function as a collagen-specific molecular chaperone (Nagata, K. (1996) Trends Biochem. Sci. 21, 23-26). However, structural requirements for substrate recognition and regulation of the binding have not yet been elucidated. Here, we show that a typical collagen model sequence, (Pro-Pro-Gly) n , possesses sufficient structural information required for recognition by HSP47. A structure-activity relationship study using synthetic analogs of (Pro-Pro-Gly) n has revealed the requirements in both chain length and primary structure for the interaction. The substrate recognition of HSP47 has also been shown to be similar but distinct from that of prolyl 4-hydroxylase, an ER resident enzyme. Further, it has shown that the interaction of HSP47 with the substrate peptides is abolished by prolyl 4-hydroxylation of the second Pro residues in Pro-ProGly triplets and that the fully prolyl 4-hydroxylated peptide, (Pro-Hyp-Gly) n , does not interact with HSP47. We thus have proposed a model in which HSP47 dissociates from procollagen during the process of prolyl 4-hydroxylation in the ER.Collagen is the most abundant protein in vertebrate bodies and is folded as procollagen in the endoplasmic reticulum (ER). 1 The folding of procollagen in cells is unique, as is the final structure itself. In the folding of type I collagen, for instance, three ␣ chains (two ␣1(I) and one ␣2(I)) first associate at C-terminal propeptides, and then approximately 340 repeats of the Xaa-Yaa-Gly triplet form the 300-nm length triple helix (1). In the triple helix, three left-handed polyproline type II (PP-II) helices are supercoiled (2). The helix-forming process is believed to be coupled with prolyl 4-hydroxylation at Yaa positions (3). HSP47 transiently interacts with procollagen molecules in the ER in the process of folding and/or modification (4, 5). HSP47 is not secreted with procollagen from the cells, because it possesses the ER retention signal, Arg-Asp-Glu-Leu, at the carboxyl end (5, 6). The expression of HSP47 is well correlated with that of various types of collagen in cultured cells, in various tissues during development, and in pathophysiological conditions (7-12). Thus HSP47 is assumed to be a collagen-specific molecular chaperone (13). However, the binding sites or motif in the procollagen molecule have not been well elucidated. The molecular basis of HSP47 function, including the regulation of the substrate association and dissociation, also remains an enigma.Here, we studied substrate recognition of HSP47 using the recombinant chaperone protein and synthetic collagen model peptides and found that a very simple collagen model peptide mimics the native substrate of HSP47. The difference in substrate recognition between HSP47 and prolyl 4-hydroxylase (P4-H) was discussed in both primary and secondary s...

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Section: Resultsmentioning

confidence: 99%

Substrate Recognition of Collagen-specific Molecular Chaperone HSP47

Koide¹,

Asada²,

Nagata³

1999

Journal of Biological Chemistry

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“…generated only when the number of triplet reiteration is large enough (26). Another series of oligopeptides, (PPG) n , were known to have a triple-helical conformation when n is sufficiently large (27). When n increased, the binding of S3 increased, and it seemed to reach a maximum when n ϭ 8.…”

Section: Binding Of the Recombinant Colg To The Peptide Substrate-mentioning

confidence: 99%

Substrate Recognition by the Collagen-binding Domain ofClostridium histolyticum Class I Collagenase

Matsushita¹,

Koide

Kobayashi

et al. 2001

Journal of Biological Chemistry

108

112

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Clostridium histolyticum type I collagenase (ColG) has a segmental structure, S1؉S2؉S3a؉S3b. S3a and S3b bound to insoluble collagen, but S2 did not, thus indicating that S3 forms a collagen-binding domain (CBD). Because S3a؉S3b showed the most efficient binding to substrate, cooperative binding by both domains was suggested for the enzyme. Monomeric (S3b) and tandem (S3a؉S3b) CBDs bound to atelocollagen, which contains only the collagenous region. However, they did not bind to telopeptides immobilized on Sepharose beads. These results suggested that the binding site(s) for the CBD is(are) present in the collagenous region. The CBD bound to immobilized collagenous peptides, (Pro-HypGly) n and (Pro-Pro-Gly) n , only when n is large enough to allow the peptides to have a triple-helical conformation. They did not bind to various peptides with similar amino acid sequences or to gelatin, which lacks a triplehelical conformation. The CBD did not bind to immobilized Glc-Gal disaccharide, which is attached to the side chains of hydroxylysine residues in the collagenous region. These observations suggested that the CBD specifically recognizes the triple-helical conformation made by three polypeptide chains in the collagenous region.Collagens are the major components of the extracellular matrix. They are the most abundant proteins in mammals, constituting a quarter of their total weight. Collagens are not only structural proteins with a high tensile strength but also affect cell differentiation, migration, and attachment. Nineteen different types are known to date, and type I collagen is the major species in higher vertebrates. In the endoplasmic reticulum of fibroblasts, collagen precursor peptides are hydroxylated by various dioxygenases (prolyl hydroxylases and a lysyl hydroxylase), glycosylated at the hydroxylysine residues, and folded into a triple helix. HSP47 associates with procollagen during this modification and/or folding processes and is assumed to function as a collagen-specific molecular chaperone (1). The precursor is secreted into the extracellular space, and its N-and C-terminal propeptides are removed by procollagen peptidases. Cleavage triggers the arrangement of collagen monomers into a staggered array called fibrils, which are insoluble macromolecular assemblies having lengths up to a few hundred micrometers. One can observe 67-nm-wide cross striations (overlap and gap zones) on the fibrils by electron microscopy, which are formed by the regular arrangement of collagen monomers. Each collagen monomer consists of collagenous and noncollagenous regions. The former have a regular amino acid sequence, which is a repeat (338 times in type I collagen) of the Gly-X-Y triplet, forming a long (300 nm in type I collagen) triple-helical domain. Proline and hydroxyproline residues are most common in the X and Y positions, respectively, and this combination stabilizes the helix to the highest extent (2). The latter regions lack the sequence and conformation characteristic of the former, and are named telopeptides,...

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“…Polypeptides [1] have been well investigated as protein model compounds [1][2][3][4][5][6][7][8]. Numerous reports on the methodology for the synthesis of polypeptides have been published [1][2][3][4][5][6][7][8][9][10][11][12][13][14].…”

Section: Introductionmentioning

confidence: 99%

“…Numerous reports on the methodology for the synthesis of polypeptides have been published [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. The N-carboxyl--amino acid anhydride (NCA) method (1) [2,3,9,10], polymerization of amino acids using active esters (2) [1,[4][5][6][7], solid-phase peptide synthesis (3) [8,12], and the heating of amino acids (4) [13,14] are typical examples. The NCA method (1) is suitable for making homopolypeptides and random copolypeptides but is not suitable for the synthesis of sequential copolyamino acid, which is more important for the build-up of functional polypeptides.…”

Section: Introductionmentioning

confidence: 99%

“…Sequential polyamino acids have repetitive amino acid residues, in which the amino acid residues can be like -(Gly-Gly-Asp) -. The active ester method (2) [1,[4][5][6][7] and solid phase synthesis (3) [8] are more suitable for the synthesis of sequential copolyamino acid. However, the problems of methods (2) and (3) are a long reaction time and the use of much solvent.…”

Section: Introductionmentioning

confidence: 99%

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Thermal Synthesis of Polypeptides from N-Butyloxycarbonyl Oligopeptides Containing Aspartyl Residue at C-Terminus

Munegumi

Yamada

2017

International Journal of Polymer Science

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The thermal reactions of amino acids have been investigated for pure organic synthesis, materials preparation in industry, and prebiotic chemistry. N-t-Butyloxycarbonyl aspartic acid (Boc-Asp) releases 2-butene and carbon dioxide upon heating without solvents. The resulting mixture of the free molten aspartic acid was dehydrated to give peptide bonds. This study describes the thermal reactions of N-t-butyloxycarbonyl peptides (Boc-Gly-L-Asp, Boc-L-Ala-L-Asp, Boc-L-Val-L-Asp, and Boc-Gly-Gly-L-Asp) having an aspartic residue at the carboxyl terminus. The peptides were deprotected upon heating at a constant temperature between 110 and 170°C for 1 to 24 h to afford polypeptides in which the average molecular weight reached 7800.

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Synthesis of Poly-(l-prolyl-l-prolylglycyl) of Defined Molecular Weights

Cited by 149 publications

References 6 publications

Substrate Recognition of Collagen-specific Molecular Chaperone HSP47

Substrate Recognition of Collagen-specific Molecular Chaperone HSP47

Substrate Recognition by the Collagen-binding Domain ofClostridium histolyticum Class I Collagenase

Thermal Synthesis of Polypeptides from N-Butyloxycarbonyl Oligopeptides Containing Aspartyl Residue at C-Terminus

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