Structural characterization of very high density lipoprotein from the spider Polybetes pythagoricus

LAINO A.; GARCIA F.; CUNNINGHAM M.; HERAS H.

Carlos Paz, Cordoba, Argentina

Congreso; XLIV Reunion Anual de la Sociedad Argentina de Investigaciones en Bioquimica y Biologia Molecular.; 2008

We have previously isolated, purificated and characterized an hemolymphatic VHDL from Polybetes pythagoricus. This lipoprotein contains hemocyanin (Hc) as the major apoprotein, and as a novelty, it transports most of the circulating lipids. In the present work, for the first time in arachnids, we studied size, shape and structure of  P. pythagoricus VHDL using electronic microscopy, MALDI-TOF, circular dichroism, partial proteolysis,  N-terminal sequenciation, and lipid and copper gel specific staining. Results showed that VHDL has spheroidal morphology with an estimated size of 11.2 ?} 0.025 nm. As seen by SDS-PAGGE, it contains one main band corresponding to monomers of Hc (70 kDa), and two minor subunits corresponding to non-respiratory proteins of 105 and 120 kDa. These last two proteins are not linked by disulfide bonds, they don?Lt include lipids or copper in their structure, and they would be more exposed to the aqueous medium in native conditions. Using circular dichroism we observed that it contains 20% a-helix, 29% ?À-sheet, 22.7% turns and 29.7% random. By MALDI-TOF, 15 polypeptides present in monomer were found to be homologous to subunit 3 of the spider Cupiennius salei hemocyanin; and 27 polypeptides from the 105 kDa subunit showed homology to a protein from the insect Anopheles gambiae.