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 ␣1 and ␣2 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 ␣1 1031 . We demonstrated that a synthetic nonapeptide mimicking the ␣1 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 ␣1 chain plays a crucial role in stipulating collagen suprastructure and in subsequent fibril formation.Type I collagen is the most abundant protein in connective tissue of all vertebrates. It forms highly ordered fibrils that are generally thought to provide mechanical strength and regulate cell function (1-5). Collagen is formed from tightly interwoven heterotrimers of two ␣1 chains and one ␣2 chain in a triplehelical coiled-coil structure. The triple helix consisting of GlyXaa-Yaa repeats is ϳ1,000 amino acid residues in length and is resistant to proteolysis except for specific collagenases that can cleave the helix (6, 7). The high content of 4-hydroxyproline in the Yaa position stabilizes the triple-helical structure (8, 9).Observations of in vitro collagen fibril formation suggest that the collagen molecule has sufficient structural information to assemble spontaneously under suitable conditions. This structural information has been partially revealed by atomic force microscopy, electron microscopy, spectroscopic analysis, and x-ray diffraction (10 -15). From this information, it appears that the telopeptide regions play an important role in the rate of fibril formation and in azimuthal and lateral growth of fibrils. Indeed, fibril formation of pepsin-hydrolyzed collagen (PHCol) 2 that has been partially hydrolyzed at the N-and C-telopeptide domains progresses more slowly than that of acidsoluble collagen (ASCol) (16,17). The recognition and association of collagen N-and C-telopeptides have been partly demonstrated in vitro (18 -25). The results imply that the Nand C-telopeptide regions are required to "seed" the interaction between collagen molecules. It was also concluded that the triple-helical structure is essential for fibril formation (4, 26).Our experimental approach to understand the mechanism of fibril formation was to investigate type I collagen that had been partially hydro...

Comparison of the Structural Characteristics of Native Collagen Fibrils Derived from Bovine Tendons Using Two Different Methods: Modified Acid-Solubilized and Pepsin-Aided Extraction

“…The N-H stretching vibrations of A-CF and P-CF separately appeared at 3423.76 cm −1 and 3324.57 cm −1 , both cases with high peak intensity. There was a relatively weak absorption peak in both A-CF and P-CF at 2957.13 cm −1 and 2934.59 cm −1 , respectively, because of the C-N stretching vibration named Amide B [32]; that is consistent with that recorded by Kunii, et al [33]. Amide I and Amide II bands of collagen were associated with α-helix, β-folding, random crimp coil and superposition.…”

Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions

“…It has previously been shown that Act d 1 exerted proteolytic activity under simulated gastric and intestinal conditions [16], and the contribution of Act d 1 to the digestion of several food proteins was confirmed in vivo [31]. In our study, an increased intestinal permeability to FITC-dextran was detected in mice which had been given [4][5][6][7][8][9][10] and wide substrate specificity [32], including collagenolytic activity [33]. Apart from contributing to the disruption of tight junctions, the collagenolytic activity of Act d 1 can lead to connective tissue loosening in the lamina propria.…”

Thermal Memory in Self-Assembled Collagen Fibril Networks

“…Bovine dermal collagen I (NUTRAGEN) was purchased as a 6.0 mg/ml solution in 0.01 M HCl from Advanced Biomatrix (San Diego, CA). This collagen is isolated from bovine hides by enzymatic digestion with pepsin, Collagen Self-Assembly Kinetics which removes portions from the N-and C-terminal telopeptides containing the lysine groups that are responsible for intermolecular cross-linking in vivo (53). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4% gels was used to assess the purity and degree of intramolecular cross-linking.…”