Hemocyanin of the Molluscan Concholepas concholepas Exhibits an Unusual Heterodecameric Array of Subunits

Abstract

We describe here the structure of the hemocyanin from the Chilean gastropod Concholepas concholepas (CCH), emphasizing some attributes that make it interesting among molluscan hemocyanins. CCH exhibits a predominant didecameric structure as revealed by electron microscopy and a size of 8 MDa by gel filtration, and, in contrast with other mollusc hemocyanins, its stabilization does not require additional Ca 2؉ and/or Mg 2؉ in the medium. Polyacrylamide gel electrophoresis studies, analyses by a MonoQ FPLC column, and Western blots with specific monoclonal antibodies showed that CCH is made by two subunits noncovalently linked, named CCH-A and CCH-B, with molecular masses of 405 and 350 kDa, respectively. Interestingly, one of the subunits undergoes changes within the macromolecule; we demonstrated that CCH-A has an autocleavage site that under reducing conditions is cleaved to yield two polypeptides, CCH-A1 (300 kDa) and CCH-A2 (108 kDa), whereas CCH-B remains unchanged. The CCH-A nick occurs at 4°C, increases at 37°C, and is not inhibited by the addition of protease inhibitors and/or divalent cations. Since the CCH structure is a heterodimer, we investigated whether subunits would be either intermingled, forming heterodecamers, or assembled as two homogeneous decamers. Light scattering and electron microscope studies of the in vitro reassociation of purified CCH subunits demonstrated that the sole addition of Mg 2؉ is needed for its reassembly into the native decameric molecule; no homodecamer reorganization was found with either CCH-A or CCH-B subunits alone. Our evidence showed that C. concholepas hemocyanin is an unusual example of heterodecameric organization.

Enhanced structural stability of Concholepas hemocyanin increases its immunogenicity and maintains its non‐specific immunostimulatory effects

“…These results were confirmed with TEM. Because of the large size of CCH, which is approximately 325Å in diameter and 392Å in height, and because of its peculiar structure as a hollow cylinder [7,8], we were able to unequivocally identify the presence of hemocyanin molecules ( Fig. 2B and C).…”

“…Several hemocyanins from other species of mollusks have been studied, including Haliotis tuberculata [5], Rapana thomasiana [6], and Concholepas concholepas hemocyanin (CCH) [7]. Only CCH has been preclinically evaluated in a murine experimental model of SBC and may be considered as an alternative therapy for SBC [8,9].…”

“…Despite KLH and CCH have different sources and quaternary structures, both proteins display similar immunogenicity and antitumor capability, which suggest that a conserved pattern induces an ancient immunogenic and immunostimulatory mechanism [7,8]. Many authors have proposed different factors to explain these properties, such as the large size, the complex quaternary structure with D5-like symmetry, the xenogenicity, and the sugar moiety content of hemocyanins.…”

TAPCells, the Chilean dendritic cell vaccine against melanoma and prostate cancer

“…In the melanoma studies it was shown that Keyhole Limpet hemocyanin is a potent inducer of immunogenicity (López et al, 2009;Aguilera et al, 2011). In the prostate studies, the Chilean Concholepas concholepas hemocyanin (CCH) (De Ioannes et al, 2004) was used for the fi rst time as an adjuvant in a vaccine against cancer. In this clinical study, the CCH was able to induce an immune memory response as measured by the DTH test, and was found to generate no toxic or allergic reactions associated with its subcutaneous administration.…”

Anti-prion activity generated by a novel vaccine formulation

“…All peptides, except peptide 4, were synthesized with either an N-terminal or C-terminal linker containing a glycine cystine for conjugation to blue carrier protein (BCP) (BioSonda Santiago, Chile) using standard sulfosuccimnimidyl 4-(N- [14]. Blue carrier protein was selected over the traditional Keyhole Limpet Hemocyanin (KLH) due to its reported improved stability, high molecular weight, and enhanced immunogenicity [3]. All vaccine conjugates were made into a water in oil emulsion using the adjuvant Adjuvac TM , developed at the National Wildlife Research Center.…”