Serum prostate-specific antigen (PSA) assay is widely used for detection of prostate cancer. Because PSA is also synthesized from normal prostate, false positive diagnosis cannot be avoided by the conventional serum PSA test. To apply the cancer-associated carbohydrate alteration to the improvement of PSA assay, we first elucidated the structures of PSA purified from human seminal fluid. The predominant core structure of N-glycans of seminal fluid PSA was a complex type biantennary oligosaccharide and was consistent with the structure reported previously. However, we found the sialic acid alpha2-3 galactose linkage as an additional terminal carbohydrate structure on seminal fluid PSA. We then analyzed the carbohydrate moiety of serum PSA from the patients with prostate cancer and benign prostate hypertrophy using lectin affinity chromatography. Lectin binding was assessed by lectin affinity column chromatography followed by determining the amount of total and free PSA. Concanavalin A, Lens culinaris, Aleuria aurantia, Sambucus nigra, and Maackia amurensis lectins were tested for their binding to the carbohydrates on PSA. Among the lectins examined, the M. amurensis agglutinin-bound fraction of free serum PSA is increased in prostate cancer patients compared to benign prostate hypertrophy patients. The binding of PSA to M. amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, was also confirmed by surface plasmon resonance analysis. These results suggest that the differential binding of free serum PSA to M. amurensis agglutinin lectin between prostate cancer and benign prostate hypertrophy could be a potential measure for diagnosis of prostate cancer.
Frogs have unique pyrimidine base-specific RNases, with structures similar to those of turtle, iguana and chicken RNases. Among the four frog RNases discussed here, three from Rana pipiens, R. catesbeiana and R. japonica oocyte cells show antitumour activity, and the latter two show lectin activity towards sialic acid-rich glycoproteins. In this review, (i) we compare their unique primary structures with respect to the locations of their disulphide bridges, three-dimensional structure, base specificity and heat stability as compared with RNase A, and (ii) we summarize current knowledge about the mode of action of lectin and the antitumour activities of the three frog RNases.
Background: Studies on the diversity of carbohydrate-binding proteins (lectins) are important in glycobiology. Results: A lectin having a novel primary structure was isolated from a mussel and found to have a globotriose-dependent cytotoxicity on Burkitt lymphoma cells. Conclusion: A new primary structure quite distinct from known lectin is described. Significance: Discovery of similar lectin structures from vertebrates will lead to progress in medical sciences.
The complete amino acid sequence of sialic acid binding lectin from frog (Rana catesbeiana) egg is presented. The 111-residue sequence was determined by the analysis of peptides generated by digestion of the S-carboxymethylated protein with Achromobacter protease I, chymotrypsin, or cyanogen bromide. The sequence is unique and not homologous to any known protein sequence. The protein may represent a new type of lectin.
A cytometrical image analyzing method for nuclear protein was established using WinROOF, a commercially available, inexpensive software, to determine the status of both estrogen and progesterone receptors. Immunohistochemical evaluation of estrogen receptors (ER) and progesterone receptors (PR) was performed with the anti-ER (clone 1D5) and the anti-PR (clone PgR636), respectively, combined with dextran polymer reagent EnVision+, all of which are approved in vitro diagnostics in Japan. The immunostained results were captured as digital images in Windows, and then analyzed in WinROOF with macroinstructions for analyzing each captured area either immunolabeled with chromogen or counterstained with hematoxylin. This image analysis method graded the immunostained nuclei of carcinoma cells based on staining intensities, and calculated the labeling index (LI) for both ER and PR. Furthermore, the LI correlated highly with the results from a histology score (HSCORE) when 20 breast carcinomas were quantified. Regarding ER, when 20% in the LI was considered as the cut-off point for positive, the positivity of ER in computer-assisted analysis was 75% (15 of 20 cases), and was completely concordant with that of HSCORE-based analysis. These results indicate that the cytometrical image analysis-based quantification could be appropriately applied to the objective determination of the immunohistochemical status of both ER and PR.
Two lectins with RNase activity obtained from eggs of Rana catesbeiana and R. japonica and RNase obtained from R. catesbeiana liver show 65-83% protein homology. The base specificity of these frog proteins was studied with 8 dinucleoside phosphates as substrates and 8 nucleotides as inhibitors. The base specificities of the B1 and B2 sites of these proteins are U greater than C and G greater than U greater than A, C, respectively. The three frog proteins are more resistant than RNase A to heat treatment, guanidine-HCl and pH-induced denaturation; i.e., they retain their native conformation up to at least 70 degrees C at pH 7.5. Differences in stability and base specificity among RNase A and the three frog proteins are discussed in relation to the primary structures. Although the two lectins agglutinate tumor cells (e.g., Ehrlich, S-180 and AH109A ascites carcinoma cells), the liver RNase has no such activity. Agglutination of AH109A cells by the two lectins is inhibited by nucleotides. Our results indicate that the agglutination sites are not identical with, but are related to, the active sites of the three frog proteins.
Sialic acid binding lectin (SBL) isolated from Rana catesbeiana oocytes is a multifunctional protein which has lectin activity, ribonuclease activity and antitumor activity. However, the mechanism of antitumor effects of SBL is unclear to date and the validity for human leukemia cells has not been fully studied. We report here that SBL shows cytotoxicity for some human leukemia cell lines including multidrug-resistant (MDR) cells. The precise mechanisms of SBL-induced apoptotic signals were analyzed by combinational usage of specific caspase inhibitors and the mitochondrial membrane depolarization detector JC-1. It was demonstrated that SBL causes mitochondrial perturbation and the apoptotic signal is amplified by caspases and cell death is executed in a caspase-dependent manner. The efficacy of this combinational usage was shown for the first time, to distinguish the apoptotic pathway in detail. SBL selectively kills tumor cells, is able to exhibit cytotoxicity regardless of P-glycoprotein expression and has potential as an alternative to conventional DNA-damaging anticancer drugs.
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