No abstract

We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and complementary DNA (cDNA) cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per liter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48 - 1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

Sialic acid represents a critical sugar component located at the outermost position of glycoconjugates, playing important roles in extensive biological processes. To date, however, there have been only few probes which show affinity to α(2,3)-linked sialic acid-containing glycoconjugates. Agrocybe cylindracea galectin is known to have a relatively high affinity towards Neu5Acα(2,3)Galβ(1,4)Glc (3'-sialyl lactose), but it significantly recognizes various β-galactosides, such as Galβ(1,4)GlcNAcβ (LacNAc) and Galβ(1,3)GalNAcα (T-antigen). To eliminate this background specificity, we focused an acidic amino acid residue (Glu86), which interacts with the glucose unit of 3'-sialyl lactose and substituted it with all other amino acids. Carbohydrate-binding specificity of the derived 14 mutants was analysed by surface plasmon resonance, and it was found that E86D mutant (Glu86 substituted with Asp) substantially lost the binding ability to LacNAc and T-antigen, while it retained the high affinity for 3'-sialyl lactose. Further, frontal affinity chromatography analysis using 132 pyridylaminated oligosaccharides confirmed that the E86D mutant had a strong preference for α(2,3)-disialo biantennary N-linked glycan. However, it showed the large decrease in the affinity for any of the asialo complex-type N-glycans and the glycolipid-type glycans. Thus, the developed mutant E86D will be of practical use in various fields relevant to cell biology and glycotechnology.

We have developed a novel method for the preparation of 'recombinant proteoliposomes'. Membrane proteins were expressed on budded virus (BV) envelopes using baculovirus gene expression systems, and proteoliposomes were prepared by fusion of these viruses with liposomes. First, plasmid DNA containing the gene for the thyroid-stimulating hormone receptor (TSHR) or the acetylcholine receptor alpha-subunit (AChRalpha) was co-transfected with wild type virus [Autographa californica nuclear polyhedrosis virus (AcNPV)] genomes into insect cells [Spodoptera frugiperda (Sf9)] to obtain recombinant viruses via homologous recombination. The recombinant viruses were again infected into Sf9 cells, and the resulting BVs were shown to express TSHR and AChRalpha. Next, the fusion behaviour of AcNPV-derived BVs and liposomes was examined via a fluorescence assay, and BVs were shown to fuse with phosphatidylserine-containing liposomes below pH 5.0, the pH at which fusion glycoprotein gp64 on the virus envelope becomes active. TSHR- or AChRalpha-expressed BVs were also shown to fuse with liposomes. Finally, TSHR- and AChRalpha-recombinant proteoliposomes were immobilized on enzyme-linked immunosorbent assay plates, and their reactivities were examined via a general immunoassay, which showed that the recombinant proteoliposomes were fully active. These results successfully demonstrate the development of a method based on a baculovirus gene expression system for the preparation of recombinant and functional proteoliposomes.

The fuzzy rat, a genetic mutant between hairless and hairy albino rats, expresses androgen-dependent hypersecretion of sebum and hyperplastic sebaceous glands. Using this model for human acne, we examined the effects of inhibitors of human steroid 5α-reductase isozymes, type I (MK386) and type II (finasteride), and an androgen receptor blocker (RU58841) on regression of glandular and ductal hyperplasia. The above three agents, 1 % weight volume, were dissolved into the vehicle (propy-lene glycol, alcohol and water) and applied on the backs of peripubertal male rats for 2 months. Control and castrate groups received vehicle alone. At 8 weeks, we examined the size of the sebaceous glandular lobules and ducts in split epidermal preparations as well as in frozen sections of skin stained with osmium-potassium dichromate solution. The number of bro-modeoxyuridine (BrdU)-positive cells was counted in the glandular lobes in split-skin tissues stained with BrdU immunochemistry. The results revealed that the sizes of both lobes and ducts in castrates were 40-60% smaller than in controls. RU58841 induced glandular and ductal regression equivalent to that in castrates. Finasteride induced a moderate degree of lobular and ductal reduction, whereas MK386 caused only ductal regression. Reduction of BrdU-positive cells in the sebaceous lobes was found in the skin treated with finasteride and RU58841. Serum concentrations of testosterone and dihydrotestosterone showed no significant changes in all drug-treated rats. The weight of the prostatic lobes was reduced significantly in rats treated with finasteride but not by the other two agents. RU58841 effectively counteracted endogenous androgens resulting in a suppression of growth of the sebaceous glands but not the prostate. This rodent model for androgen-dependent hyperplasia of the sebaceous glands is useful for the study of many pharmacological aspects comprising the rate of percutaneous absorption, stability and affinity to target organs of the testing compounds, and selection of adequate vehicles for topical application.