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Actinidain-hydrolyzed type I collagen reveals a crucial amino acid sequence in fibril formation.

ABSTRACT: We investigated the ability of type I collagen telopeptides to bind neighboring collagen molecules, which is thought to be the initial event in fibrillogenesis. Limited hydrolysis by actinidain protease produced monomeric collagen, which consisted almost entirely of alpha1 and alpha2 chains. As seen with ultrahigh resolution scanning electron microscopy, actinidain-hydrolyzed collagen exhibited unique self-assembly, as if at an intermediate stage, and formed a novel suprastructure characterized by poor fibrillogenesis. Then, the N- and C-terminal sequences of chicken type I collagen hydrolyzed by actinidain or pepsin were determined by Edman degradation and de novo sequence analysis with matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry, respectively. In the C-telopeptide region of the alpha1 chain, pepsin cleaved between Asp(1035) and Phe(1036), and actinidain between Gly(1032) and Gly(1033). Thus, the actinidain-hydrolyzed alpha1 chain is shorter at the C terminus by three residues, Gly(1033), Phe(1034), and Asp(1035). In the alpha2 chain, both proteases cleaved between Glu(1030) and Val(1031). We demonstrated that a synthetic nonapeptide mimicking the alpha1 C-terminal sequence including GFD weakly inhibited the self-assembly of pepsin-hydrolyzed collagen, whereas it remarkably accelerated that of actinidain-hydrolyzed collagen. We conclude that the specific GFD sequence of the C-telopeptide of the alpha1 chain plays a crucial role in stipulating collagen suprastructure and in subsequent fibril formation.

SUBMITTER: Kunii S

PROVIDER: S-EPMC2878510 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Actinidain-hydrolyzed type I collagen reveals a crucial amino acid sequence in fibril formation.

Kunii Saori S Morimoto Koichi K Nagai Kouhei K Saito Takuya T Sato Kenji K Tonomura Ben'ichiro B

The Journal of biological chemistry 20100414 23

We investigated the ability of type I collagen telopeptides to bind neighboring collagen molecules, which is thought to be the initial event in fibrillogenesis. Limited hydrolysis by actinidain protease produced monomeric collagen, which consisted almost entirely of alpha1 and alpha2 chains. As seen with ultrahigh resolution scanning electron microscopy, actinidain-hydrolyzed collagen exhibited unique self-assembly, as if at an intermediate stage, and formed a novel suprastructure characterized ...[more]

PMID: 20392700

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Project description:1. The effects of acid mucopolysaccharides and acid mucopolysaccharide-proteins on the size and rate of formation of fibril aggregates from collagen solutions in pH7.6 buffers were studied by turbidimetric and light-scattering methods. 2. Serum albumin, orosomucoid, methylated cellulose, chondroitin sulphate A and chondroitin sulphate C of molecular weight less than 20000, and hyaluronate of molecular weight less than 40000 did not influence rates of fibril formation. Chondroitin sulphate A, chondroitin sulphate C and hyaluronate of high molecular weight retarded the rate of fibril formation. This effect of high-molecular-weight chondroitin sulphate C decreased with increasing ionic strength. Heparin, though of low molecular weight (13000), was highly effective, as was also heparitin sulphate. The chondroitin sulphate-proteins of very high molecular weight were highly effective, despite the fact that for some preparations the component chondroitin sulphate chains had molecular weights much less than 20000. 3. Agents that had delayed fibril formation were also effective in producing an increase in degree of aggregation of fibrillar collagen, as indicated by dissymmetry changes observed in light-scattering experiments at low collagen concentrations. Methylated cellulose and heparin at 2.5mug./ml. were unusual in decreasing aggregation, but heparin at 0.25mug./ml. increased aggregation. Electron microscopy of gels showed fibrils and fibril aggregates with ;normal' collagen spacing and dimensions consistent with the light-scattering results. 4. The rates of electrical transport of agents and of solvent (electro-osmosis) through collagen gels indicated a contribution of molecular entanglement that increased with increase in molecular size of the agents. Electrostatic binding of heparin to collagen was noted. Binding to collagen during fibril formation was also found for heparitin sulphate and a chondroitin sulphate with extra sulphate groups. 5. Electrostatic binding of acid mucopolysaccharide-proteins to collagen may be an important factor in the organization and functioning of connective tissues at all stages of growth and development. Excluded-volume (molecular-entanglement) effects may also be important. These factors operate simultaneously and interact mutually so that precise assessment of their relative importance is difficult.

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Actinidain-hydrolyzed type I collagen reveals a crucial amino acid sequence in fibril formation.

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Actinidain-hydrolyzed type I collagen reveals a crucial amino acid sequence in fibril formation.

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