Immunological Potential of Helix vulgaris and Rapana venosa Hemocyanins

A new hemocyanin was isolated from the hemolymph of garden snails Helix vulgaris, composed of two isoforms, HvH1 and HvH2 separated on an ion exchange column DEAE-Sepharose 6CL. Structural and immunological properties of Helix vulgaris hemocyanin were studied in comparison with molluscan Hcs Rapana venosa and Megathura crenulata. The possibility of using HvH and RvH as carriers of small molecules (haptens) in immunizing protocols was studied in comparison with KLH, which is a widely used, highly immunogenic carrier protein. By using HvH as a carrier of the well-known hapten TNBS (2,4,6-trinitrobenzene sulfonic acid), an increasing with time production of hapten-specific TFN-γ was detected in splenocyte cultures of mice, which lasted longer than in case of KLH and RvH carriers. Also, use of HvH or RvH as a carrier of the hapten ProTα[101–109] (i.e., the synthetic C-terminal fragment of the poorly immunogenic protein prothymosin alpha) showed that antisera of higher titres than that of the control conjugate (ProTα[101–109]-KLH) were obtained immediately after the second bleeding. HvH and RvH may prove to be useful for the development of new antiviral, antibacterial and antitumor vaccines, since they seem to launch strong and specific immune response against the conjugated antigens.

[1]  S. Stevanović,et al.  Oligosaccharide structure of a functional unit RvH1-b of Rapana venosa hemocyanin using HPLC/electrospray ionization mass spectrometry. , 2007, Biochimie.

[2]  S. Stevanović,et al.  A challenging insight on the structural unit 1 of molluscan Rapana venosa hemocyanin. , 2007, Archives of biochemistry and biophysics.

[3]  J. V. Van Beeumen,et al.  New insights in Rapana venosa hemocyanin N-glycosylation resulting from on-line mass spectrometric analyses. , 2007, Glycobiology.

[4]  J. Markl,et al.  The First Complete cDNA Sequence of the Hemocyanin from a Bivalve, the Protobranch Nucula nucleus , 2007, Journal of Molecular Evolution.

[5]  P. De Ioannes,et al.  Immunotherapeutic effect of Concholepas hemocyanin in the murine bladder cancer model: evidence for conserved antitumor properties among hemocyanins. , 2006, The Journal of urology.

[6]  O. Tsitsilonis,et al.  Development and immunochemical evaluation of antibodies Y for the poorly immunogenic polypeptide prothymosin alpha , 2006, Peptides.

[7]  R. Toshkova,et al.  Protective effect of Rapana venosa hemocyanin (RvH) on survivability of hamsters with transplanted myeloid Graffi tumours , 2006 .

[8]  J. Markl,et al.  The Hemocyanin from a Living Fossil, the Cephalopod Nautilus pompilius: Protein Structure, Gene Organization, and Evolution , 2006, Journal of Molecular Evolution.

[9]  R. Kerns,et al.  Identification of DHBcAg as a potent carrier protein comparable to KLH for augmenting MUC1 antigenicity. , 2005, Vaccine.

[10]  S. Stevanović,et al.  Structure of hemocyanin subunit CaeSS2 of the crustacean Mediterranean crab Carcinus aestuarii. , 2005, Journal of biochemistry.

[11]  A. Deelder,et al.  A novel Gal(beta1-4)Gal(beta1-4)Fuc(alpha1-6)-core modification attached to the proximal N-acetylglucosamine of keyhole limpet haemocyanin (KLH) N-glycans. , 2004, The Biochemical journal.

[12]  C. Gielens,et al.  Glycosylation sites of hemocyanins of Helix pomatia and Sepia officinalis. , 2004, Micron.

[13]  S. Stevanović,et al.  Carbohydrate moieties of molluscan Rapana venosa hemocyanin. , 2004, Micron.

[14]  S. Stevanović,et al.  Characterization of the carbohydrate moieties of the functional unit RvH1-a of Rapana venosa haemocyanin using HPLC/electrospray ionization MS and glycosidase digestion. , 2003, The Biochemical journal.

[15]  S. Stevanović,et al.  Oligomeric stability of Rapana venosa hemocyanin (RvH) and its structural subunits. , 2003, Biochimica et biophysica acta.

[16]  E. Hannappel,et al.  The thymosins. Prothymosin alpha, parathymosin, and beta-thymosins: structure and function. , 2003, Vitamins and hormones.

[17]  J. Markl,et al.  Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N-glycans with Gal(beta1-6)Man-motifs. , 2002, European journal of biochemistry.

[18]  A. Evstafieva,et al.  Sensing prothymosin alpha origin, mutations and conformation with monoclonal antibodies. , 2002, Journal of immunological methods.

[19]  J. Markl,et al.  Complete hemocyanin subunit sequences of the hunting spider Cupiennius salei: recent hemocyanin remodeling in entelegyne spiders. , 2002, The Journal of biological chemistry.

[20]  S. Endres,et al.  Tumor cell lysate-pulsed human dendritic cells induce a T-cell response against pancreatic carcinoma cells: an in vitro model for the assessment of tumor vaccines. , 2001, Cancer research.

[21]  R. Abrashev,et al.  Isolation and spectroscopic characterization of the structural subunits of keyhole limpet hemocyanin. , 2001, Biochimica et biophysica acta.

[22]  S. L. Berger,et al.  Mobility Within the Nucleus and Neighboring Cytosol Is a Key Feature of Prothymosin-α , 2000, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[23]  J R Harris,et al.  Keyhole limpet hemocyanin (KLH): a biomedical review. , 1999, Micron.

[24]  D. Costopoulou,et al.  Direct ELISA method for the specific determination of prothymosin alpha in human specimens. , 1998, Journal of immunoassay.

[25]  K. V. van Holde,et al.  Sequence of the Octopus dofleini hemocyanin subunit: structural and evolutionary implications. , 1998, Journal of molecular biology.

[26]  J. Thomas-Oates,et al.  Primary structure of 21 novel monoantennary and diantennary N-linked carbohydrate chains from alphaD-hemocyanin of Helix pomatia. , 1997, European journal of biochemistry.

[27]  G. Damaschun,et al.  Prothymosin alpha: a biologically active protein with random coil conformation. , 1995, Biochemistry.

[28]  N. Boisset,et al.  Three-dimensional reconstruction of the αD and βC-hemocyanins of Helix pomatia from frozen-hydrated specimens , 1995 .

[29]  W. Voelter,et al.  A thymosin β4 ELISA using an antibody against the N terminal fragment thymosin β4[1–14] , 1992 .

[30]  M. Beltramini,et al.  Hemocyanins: molecular architecture, structure and reactivity of the binuclear copper active site. , 1990 .

[31]  C. Gielens,et al.  Identification, separation and characterization of the haemocyanin components of Helix aspersa , 1987 .

[32]  J. Vaitukaitis.  Production of antisera with small doses of immunogen: multiple intradermal injections. , 1981, Methods in enzymology.

[33]  A. Stavitsky,et al.  Immunochemical and immunogenic properties of a purified keyhold limpet haemocyanin. , 1972, Immunology.

[34]  S. Avrameas,et al.  Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. , 1969, Immunochemistry.

[35]  M. Rittenberg,et al.  Immunogenicity of trinitrophenyl-hemocyanin: production of primary and secondary anti-hapten precipitins. , 1966, Journal of immunology.