Receptor for activated C-kinase (RACK1) plays a central role in Ras-mediated signal transduction

Presented at the 16th International Charles Heidelberger Symposium on Cancer Research, Coimbra, Portugal. 26–28 September 2010Peer ReviewedpublishedVersio

Comparative analysis of IgM sub-variants in salmonid fish and identification of a residue in mu 3 which is essential for MAb4C10 reactivity

In rainbow trout (Onchorhynchus mykiss) it has been shown that high affinity IgM antibodies havea higher degree of disulfide polymerization and a longer half life time. In the present study, distinct IgMsub-variants related to ancestral tetraploidy in salmonidfish were analyzed to reveal possible charac-teristic differences between these. A monoclonal antibody (MAb4C10) which distinguishes between IgM-A and IgM-B in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) was further characterized. Itwas shown that substitution of a proline located in the loop between the B and C beta strands of the thirdconstant domain (m3) of salmonmA eliminated MAb4C10 reactivity. Accordingly, the reverse substitutionin salmonmB restored MAb4C10 reactivity. Molecular cloning ofmcDNA from arctic char (Salvelinusalpinus) revealed two sub-variants (mA-1 andmA-2), i.e. a similar situation as in Atlantic salmon andbrown trout. However, arctic char IgM eluted in one peak by anion exchange chromatography, in contrastto salmon and brown trout IgM that are eluted in two peaks. The only characteristic residue of salmonand brown troutmB is an additional cysteine in the C-terminal part ofm4. Most likely, this cysteine isinvolved in inter-chain disulfide bonding and influences the elution profiles of IgM-A and IgM-B on anionexchange chromatography. Neither of themsub-variants in arctic char have the additional cysteine, andchar IgM, as well as salmon and brown trout IgM-A, showed a lower degree of inter-chain disulfidebonding than IgM-B when subjected to denaturation and gel electrophoresis under non-reducingconditions. Hybrids of char/salmon expressedmA-1,mA-2,mA andmB, indicating that there are twoparalogous Ig heavy chain gene complexes in the haploid genome of char, like in Atlantic salmon. Acomparison of salmonidmsequences is presented, including representatives ofSalmoninae(trout, salmonand char),Thymallinae(grayling) andCoregoninae(whitefish)submittedVersio

Cloning, expression and purification of Atlantic salmon (Salmo salar, L.) neuroglobin

Neuroglobin (Ngb) exists only in small amounts in salmon brain. In order to study the protein in more detail salmon neuroglobin (sNgb) was cloned, hetereologously expressed in E.coli and purified. The protein had red color and showed the characteristic peaks at 411 nm (metNgb), 415 nm (carboxyNgb) and 424 nm (deoxyNgb). Western analysis showed that sNgb reacted weakly against a rabbit anti human neuroglobin (hNgb) and strongly to a sNgb specific antibody. Our 3Dhomology model of the sNgb indicated modifications adjacent to and in the O2/CO binding site. This may correlate to differences in substrate affinities for the sNgb compared to the hNgb. Also sNgb contained shorter helixes and longer interhelical loops typical for psycrophilic proteins

Expression and purification of receptor for activated C-kinase 1 (RACK1)

International audienc

Comparative analysis of IgM sub-variants in salmonid fish and identification of a residue in mu 3 which is essential for MAb4C10 reactivity

In rainbow trout (Onchorhynchus mykiss) it has been shown that high affinity IgM antibodies havea higher degree of disulfide polymerization and a longer half life time. In the present study, distinct IgMsub-variants related to ancestral tetraploidy in salmonidfish were analyzed to reveal possible charac-teristic differences between these. A monoclonal antibody (MAb4C10) which distinguishes between IgM-A and IgM-B in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) was further characterized. Itwas shown that substitution of a proline located in the loop between the B and C beta strands of the thirdconstant domain (m3) of salmonmA eliminated MAb4C10 reactivity. Accordingly, the reverse substitutionin salmonmB restored MAb4C10 reactivity. Molecular cloning ofmcDNA from arctic char (Salvelinusalpinus) revealed two sub-variants (mA-1 andmA-2), i.e. a similar situation as in Atlantic salmon andbrown trout. However, arctic char IgM eluted in one peak by anion exchange chromatography, in contrastto salmon and brown trout IgM that are eluted in two peaks. The only characteristic residue of salmonand brown troutmB is an additional cysteine in the C-terminal part ofm4. Most likely, this cysteine isinvolved in inter-chain disulfide bonding and influences the elution profiles of IgM-A and IgM-B on anionexchange chromatography. Neither of themsub-variants in arctic char have the additional cysteine, andchar IgM, as well as salmon and brown trout IgM-A, showed a lower degree of inter-chain disulfidebonding than IgM-B when subjected to denaturation and gel electrophoresis under non-reducingconditions. Hybrids of char/salmon expressedmA-1,mA-2,mA andmB, indicating that there are twoparalogous Ig heavy chain gene complexes in the haploid genome of char, like in Atlantic salmon. Acomparison of salmonidmsequences is presented, including representatives ofSalmoninae(trout, salmonand char),Thymallinae(grayling) andCoregoninae(whitefish