Characteristics of Pepsin-Solubilised Collagen from the Skin of Splendid Squid (Loligo formosana)

Pepsin-solubilised collagen from the skin of splendid squid (SC) was isolated, partially purified by salt precipitation and dialysis prior to characterisation. The yield of SC was 75.3% (dry weight basis). SC with high purity was obtained as shown by the distinct UV absorption peak at 232 nm and high hydroxyproline content. Total sugar content of SC was 4.70% (dry weight basis), which was higher than that of collagen from calf skin (CC) (1.45% dry weight basis) (P<0.05). Based on SDS-PAGE and elution profile, SC might contain the mixed types of collagen (type SQ-I and type SQ-II), in whichα- andβ-chains were the major components. SC was rich in glycine and had high content of imino acids (189 residues/1000 residues). The degradation induced by chymotrypsin and lysyl endopeptidase was more pronounced in CC, compared with SC. The maximum transition temperature (Tmax) of SC was 34.1°C, which was about 7°C lower than that of CC. Fourier transform infrared spectra revealed that the triple-helical structure of SC was predominant with the copresence of carbohydrate moieties. Therefore, the skin of splendid squid, a byproduct from squid processing, can be an alternative source for collagen production.

Lactococcus lactis Gene yjgB Encodes a γ-d-Glutaminyl-l-Lysyl- Endopeptidase Which Hydrolyzes Peptidoglycan

ABSTRACT YjgB is one of five peptidoglycan hydrolases previously identified in Lactococcus lactis. Analysis of its amino acid sequence revealed that YjgB contains an NlpC/P60 domain, whereas no specific cell wall binding domain or motif could be identified. The NlpC/P60 family is characterized by three conserved residues, a cysteine, a histidine, and a polar residue. In agreement with the presence of a Cys residue in the catalytic site of YjgB, its enzymatic activity was enhanced in the presence of dithiothreitol. Peptidoglycan-hydrolyzing activity of YjgB was detected in growing cells of an L. lactis strain overexpressing YjgB, as revealed by the presence of disaccharide (DS)-dipeptide in the muropeptide composition of the overexpressing strain. YjgB hydrolyzes the peptide chains of L. lactis muropeptides between γ-d-Gln and l-Lys residues. Its hydrolytic activity was detected on DSs with tetra- and pentapeptide chains, whereas hydrolytic activity was very low on DS-tripeptides. Thus, we demonstrated that YjgB is an endopeptidase which cleaves γ-d-Gln-l-Lys bonds in peptide chains of L. lactis peptidoglycan.

A Second Lysine-Specific Serine Protease from Lysobacter sp. Strain IB-9374

ABSTRACT A second lysyl endopeptidase gene (lepB) was found immediately upstream of the previously isolated lepA gene encoding a highly active lysyl endopeptidase in Lysobacter genomic DNA. The lepB gene consists of 2,034 nucleotides coding for a protein of 678 amino acids. Amino acid sequence alignment between the lepA and lepB gene products (LepA and LepB) revealed that the LepB precursor protein is composed of a prepeptide (20 amino acids [aa]), a propeptide (184 aa), a mature enzyme (274 aa), and a C-terminal extension peptide (200 aa). The mature enzyme region exhibited 72% sequence identity to its LepA counterpart and conserved all essential amino acids constituting the catalytic triad and the primary determining site for lysine specificity. The lepB gene encoding the propeptide and mature-enzyme portions was overexpressed in Escherichia coli, and the inclusion body produced generated active LepB through appropriate refolding and processing. The purified enzyme, a mature 274-aa lysine-specific endopeptidase, was less active and more sensitive to both temperature and denaturation with urea, guanidine hydrochloride, or sodium dodecyl sulfate than LepA. LepA-based modeling implies that LepB can fold into essentially the same three-dimensional structure as LepA by placing a peptide segment, composed of several inserted amino acids found only in LepB, outside the molecule and that the Tyr169 side chain occupies the site in which the indole ring of Trp169, a built-in modulator for unique peptidase functions of LepA, resides. The results suggest that LepB is an isozyme of LepA and probably has a tertiary structure quite similar to it.

Glyceraldehyde-3-Phosphate Dehydrogenase of Streptococcus oralis Functions as a Coadhesin for Porphyromonas gingivalis Major Fimbriae

ABSTRACT Cohesive interactions between Porphyromonas gingivalis and plaque-forming bacteria, such as Streptococcus oralis, are considered to play an important role in the colonization of P. gingivalis in periodontal sites. Although P. gingivalis fimbriae have been reported to mediate coaggregation with S. oralis, the S. oralis molecule involved has not been identified. We identified the coadhesin of S. oralis ATCC 9811 and purified it by affinity column chromatography. We found that the molecular mass of the purified protein was approximately 40 kDa. Dot blot and Western blot assays showed binding of the 40-kDa protein to P. gingivalis fimbriae. Further, turbidimetric assays showed that the coadhesin inhibited coaggregation between P. gingivalis and S. oralis in a dose-dependent manner. Analyses of the amino-terminal sequences of the protein and its lysyl endopeptidase-cleaved fragments revealed that the coadhesin was identical to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Next, we cloned the gene that encodes S. oralis GAPDH and found that the sequence had a high degree of homology with the sequences of GAPDHs of various bacteria, including Streptococcus gordonii and Fusobacterium nucleatum. To confirm the contribution of S. oralis GAPDH to the interaction with P. gingivalis, a recombinant GAPDH protein was generated in Escherichia coli; this protein bound to P. gingivalis fimbriae and had an inhibitory effect on coaggregation. These results suggest that S. oralis GAPDH functions as a coadhesin for P. gingivalis fimbriae. In addition, considering the high degree of homology of the GAPDHs of various bacteria, those of other plaque-forming bacteria also may contribute to the colonization of P. gingivalis.