Tetsuya Tanaka

Growth-promoting effects of lactoferrin on L. acidophilus and Bifidobacterium spp.

We investigated the effects of lactoferrin on the growth of L. acidophilus CH-2, Bifidobacterium breve ATCC 15700, B. longum ATCC 15707, B. infantis ATCC 15697, and B. bifidum ATCC 15696. The growth of L. acidophilus was stimulated by bovine holo-lactoferrin but not by apo-lactoferrin. With bifidobacteria, bovine lactoferrin stimulated growth of three strains: B. breve, B. infantis and B. bifidum under certain conditions. Both apoprotein and holoprotein had similar effects. However, B. longum growth was not affected by lactoferrin. Thus, the mechanism of stimulating growth of bifidobacteria may be different from that of L. acidophilus. By far-western blotting using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin, lactoferrin-binding proteins were detected in the membrane protein fraction of L. acidophilus, B. bifidum, B. infantis and B. breve. The molecular weights of lactoferrin-binding proteins of L. acidophilus were estimated from SDS-polyacrylamide gel electrophoresis to be 27, 41 and 67 kDa, and those of the three bifidobacterial strains were estimated to be 67–69 kDa. However, no such lactoferrin-binding components were detected in the membrane fraction of B. longum. It is interesting that the appearance of lactoferrin-binding proteins in the membrane fraction of these species corresponds to their growth stimulation by lactoferrin.

Cathepsin E from rat neutrophils: its properties and possible relations to cathepsin D-like and cathepsin E-like acid proteinases.

An extract of rat neutrophils was found to contain a high hemoglobin-hydrolyzing activity at pH 3.2, about 70% of which does not cross-react with anti-rat liver cathepsin D antibody. A neutrophil non-cathepsin D acid proteinase was successfully isolated from cathepsin D and characterized in comparison with the properties of rat liver cathepsin D. The neutrophil enzyme differed from cathepsin D in chromatographic and electrophoretic behaviors as well as immunological cross-reactivity, and its molecular weight was estimated to be 98,000 by gel filtration on Toyopearl HW 55. These findings strongly suggest that the neutrophil enzyme could be classified as cathepsin E. The enzyme, now designated rat cathepsin E, had an optimal pH at 3.0-3.2, preferred hemoglobin to albumin as substrate, and was markedly resistant to urea denaturation. Rat cathepsins D and E cleaved the insulin B-chain at six and eight sites, respectively; five sites were common for both enzymes. Possible relations among cathepsin E and cathepsin D-like or E-like acid proteinases reported so far were discussed.

Antiviral activity of lactoferrin against canine herpesvirus.

Lactoferrin (LF) is an iron-binding protein that is found in milk and other mammalian secretions. We found that bovine lactoferrin (bLF) inhibited both the in vitro infection and replication of canine herpesvirus (CHV) in Madin-Darby canine kidney (MDCK) cells. Incubation of CHV with bLF prevented subsequent infection of MDCK cells. Furthermore, proteins from CHV-infected MDCK cells were resolved by SDS-PAGE, and then bLF CHV-binding proteins were identified by far Western blotting. We demonstrated that the anti-CHV activity of bLF was due to its interaction with CHV as well as with MDCK cells. Both the apo- and holo-forms of bLF inhibited virus multiplication independently of their iron-withholding properties. We also demonstrated that human LF had anti-CHV activity. Our findings suggest that LF could be effective in dogs to provide protection against CHV infection.

Immunochemical similarity between a gastric mucosa non-pepsin acid proteinase and neutrophil cathepsin E of the rat

Antiserum against a rat gastric mucosa non-pepsin acid proteinase precipitates rat neutrophil cathepsin E, with a precipitation curve essentially similar to that of the gastric enzyme. Taken together that the antiserum precipitates a cathepsin E-like acid proteinase from rat spleen (Muto, N., Yamamoto, M. and Tani, S. (1987) J. Biochem. (Tokyo) in press), the data indicate that the non-cathepsin D acid proteinases in rat neutrophils, gastric mucosa and spleen are immunochemically closely related. In contrast with the earlier data, cathepsin E from rabbit neutrophils exhibited a maximal activity at around pH 3.0-3.2 and preferred hemoglobin to albumin as substrate, which supports that the non-cathepsin D acid proteinases in the rat tissues are relevantly classified as cathepsin E.

Expression and Characterization of Bovine Lactoperoxidase by Recombinant Baculovirus

Lactoperoxidase (LPO) is a heme-containing oxidation-reduction enzyme present in milk. In this study, the gene encoding bovine lactoperoxidase (bLPO) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLPO was isolated. A bLPO-related recombinant baculovirus-expressed protein of 78 kDa was detected using anti-bLPO antibodies. After digestion with N-glycosidase F, the molecular weight of the recombinant bLPO (rbLPO) decreased. In addition, rbLPO reacted with lectin, indicating that the protein was glycosylated. The rbLPO activity and heme content in the culture supernatants increased upon addition of δ-aminolevulinic acid, which is a heme precursor. Differences in the δ-aminolevulinic acid-dependent circular dichroism spectrum and rbLPO pepsin hydrolysis were observed. These results suggest that the secondary structure and structural stability of rbLPO depends on the heme environment. Our data suggest that this bLPO expression system is useful for studying structure, catalytic mechanisms, and biological function.

Susceptibility of bovine osteopontin to chymosin

Osteopontin (OPN) is an acidic phosphorylated glycoprotein found in many tissues and physiological fluids. Bovine OPN is a mature protein comprising 262 amino acids with a calculated molecular weight of 29 kDa. However, SDS-PAGE analysis reveals that the protein isolated from milk migrates to a molecular mass of 60 kDa (Sorensen & Petersen, 1993; Bayless et al. 1997). Bovine milk OPN is phosphorylated at 27 serine residues and one threonine residue (Sorensen et al. 1995); three O-glycosylated threonines were also identified, but no asparagine residues were glycosylated in spite of the presence of three putative N-glycosylation sites. In contrast, eight phosphates are recognized in bovine bone OPN (Salih et al. 1996), and 12 phosphoserines and one phosphothreonine are proposed in addition to five O-linked oligosaccharides and at most one N-linked oligosaccharide in the case of rat bone OPN (Prince et al. 1987). Thus, the possibility of tissue or species-specific differences in post-translational modification has been discussed.

Expression and characterization of bovine lactoperoxidase by recombinant vaccinia virus

Lactoperoxidase (LPO) is a 78xa0kDa heme-containing oxidation–reduction enzyme present in milk, found in physiological fluids of mammals. LPO has an antimicrobial activity, and presumably contribute to the protective functions of milk against infectious diseases. In this study, recombinant vaccinia virus expressing bovine LPO (vv/bLPO) was constructed. In rabbit kidney (RK13) cells infected with vv/bLPO, recombinant bLPO was detected in both cell extracts and culture supernatants. Tunicamycin treatment decreased the molecular weight of recombinant bLPO, indicating that recombinant bLPO contains a N-linked glycosylation site. The replication of recombinant vaccinia viruses expressing bovine lactoferrin (vv/bLF) at a multiplicity of infection (moi) of 5 plaque-forming units (PFU)/cell was inhibited by antiviral activity of recombinant bLF, suggesting that vv/bLF has an antiviral effect against vaccinia virus. On the other hand, the replication of vv/bLPO at a moi of 5xa0PFU/cell was not inhibited by antiviral activity of recombinant bLPO, indicating that this recombinant virus could be used as a suitable viral vector. These results indicate that a combination of bLPO and vaccinia virus vector may be useful for medical and veterinary applications in vivo.