Completion of the amino acid sequence of the alpha 1 chain from type I calf skin collagen. Amino acid sequence of alpha 1(I)B8.
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ABSTRACT:
SUBMITTER: Glanville RW
PROVIDER: S-EPMC1152379 | biostudies-other | 1983 Oct
REPOSITORIES: biostudies-other
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Project description:The N-terminal extension peptide of type III procollagen, isolated from foetal-calf skin, contains 130 amino acid residues. To determine its amino acid sequence, the peptide was reduced and carboxymethylated or aminoethylated and fragmented with trypsin, Staphylococcus aureus V8 proteinase and bacterial collagenase. Pyroglutamate aminopeptidase was used to deblock the N-terminal collagenase fragment to enable amino acid sequencing. The type III collagen extension peptide is homologous to that of the alpha 1 chain of type I procollagen with respect to a three-domain structure. The N-terminal 79 amino acids, which contain ten of the 12 cysteine residues, form a compact globular domain. The next 39 amino acids are in a collagenase triplet sequence (Gly- Xaa - Yaa )n with a high hydroxyproline content. Finally, another short non-collagenous domain of 12 amino acids ends at the cleavage site for procollagen aminopeptidase, which cleaves a proline-glutamine bond. In contrast with type I procollagen, the type III procollagen extension peptides contain interchain disulphide bridges located at the C-terminus of the triple-helical domain.
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Project description:Papain was inhibited with bromo[2-(14)C]acetic acid, the tertiary structure of the inhibited enzyme was unfolded and the disulphide bridges were reduced with mercaptoethanol and aminoethylated. Digestion with trypsin gave a radioactive peptide consisting of residues 18-58 inclusive and containing therefore the sequence of the thirteen unknown residues 29-41 in the primary sequence of papain. This peptide was digested with pepsin to give a radioactive peptide consisting of residues 18-47, which after digestion with 0.4m-hydrochloric acid gave a radioactive peptide consisting of residues 24-43 inclusive. Further digestion with 6m-hydrochloric acid gave peptides that were used to determine the sequence: Ser-Ala-Val-Val-Thr-Ile-Glx-Gly-Ile-Ile-Lys-Ile-Arg for the residues 29-41, so completing the amino acid sequence of papain.
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Project description: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.
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Project description:1. Soluble calf-skin collagen has been denatured thermally between 37 degrees and 60 degrees and the component proteins have been separated on carboxymethylcellulose. 2. Four main fractions have been separated; alpha and beta (in the nomenclature in common usage) and two other fractions. (The alpha and beta components are complex owing to the presence of alpha(1), alpha(2), beta(1) and beta(2) parts). 3. Fractions 3 and 4 undergo rapid denaturation between 39 degrees and 40 degrees whereafter fraction 4 remains virtually unchanged even at 60 degrees . 4. That portion of fraction 4 which remains at 60 degrees is thought to be identical with the fraction designated gamma by other workers, this fraction being composed of three alpha-chains in covalent linkage (such bonds are alkali-labile). 5. The equilibrium between alpha, beta and fractions 3 and 4 is apparently reversible since acid-soluble collagen after denaturation at 45 degrees or 60 degrees followed by cooling to 0 degrees for 30min. was found to contain only fraction 4 when chromatographed at 37 degrees .
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Project description:The amino acid sequence of an amyloid-fibril protein Es492 of immunoglobulin-lambda-light-chain origin (AL) was elucidated. The amyloid fibrils were obtained from the spleen of a patient who died from systemic amyloidosis. The amino acid sequence was elucidated from structural studies of peptides derived from digestion of the protein with trypsin, thermolysin, chymotrypsin and Staphylococcus aureus V8 proteinase and from cleavage of the protein with CNBr and BNPS-skatole. A heterogeneity in the length of the polypeptide was seen in the C-terminal region. The protein was by sequence homology to other lambda-chains shown to be of the V lambda II subgroup. Although an extensive homology was seen, some amino acid residues in positions 26, 31, 32, 40, 44, 93, 97, 98 and 99 have not previously been reported in these positions of V lambda II proteins. The significance of these residues in the fibril formation is unclear. The protein was found to contain carbohydrate, with glycosylation sites in two of the hypervariable regions.
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