Apolipoprotein J (apoJ)-containing high-density lipoproteins (HDL), isolated from human plasma by immunoaffinity chromatography, are associated with apoAI and a protein of approximately 44 kDa. In order to advance our understanding of apoJ's role in the vasculature, a comprehensive investigation was performed to identify and characterize this 44-kDa protein and to study its interaction with apoJ. The 44-kDa protein, a monomeric glycoyslated polypeptide, was identified by N-terminal sequencing as serum paraoxonase. Paraoxonase exists in two oxidation states: one contains all free cysteines while the other has one disulfide bond between Cys42 and Cys284. Northern analysis of eight human tissues shows paraoxonase message present only in the liver. The majority of apoJ/paraoxonase-HDL are 90-140 kDa; however, not all of the plasma paraoxonase is associated with apoJ. The specificity of the apoJ/paraoxonase interaction, inferred by the constant mole ratio of the two proteins in affinity-purified apoJ-HDL, is confirmed in direct binding assays. For purified proteins, there is more than a 5-fold increase in the apparent affinity of apoJ for immobilized paraoxonase as the paraoxonase coating concentration is increased from 0.5 to 2.0 micrograms/mL. Both oxidation states of paraoxonase bind to apoJ with equal affinity. Our data combined with other evidence suggest that the plasma link of apoJ with paraoxonase will be implicated as a predictor of vascular damage.

Prosaposin is the precursor of four activator proteins, termed saposins A, B, C, and D, that are required for much of glycosphingolipid hydrolysis. The intact precursor also has neurite outgrowth activity ex vivo and in vivo that is localized to amino acid residues 22-31 of saposin C. Across species, this saposin C region has a high degree of identity and similarity with amino acids in the analogous region of saposin A. Wild-type and mutant saposins C and A from human and mouse were expressed in E. coli. Pure proteins, synthetic peptide analogues, conformation-specific antibodies, and CD spectroscopy were used to evaluate the basis of the ex vivo neuritogenic effect. Wild-type saposin A had no neuritogenic activity whereas reduced and alkylated saposin A did. Introduction of the conserved saposin A Tyr 30 (Y30) into saposin C at the analogous position 31, a conserved Ala(A)/Gly(G)31, diminished neuritogenic activity by 50-60%. Nondenatured saposin A with an introduced A30 acquired substantial neuritogenic activity. Polyclonal antibodies directed against the NH2-terminus of saposin C cross-reacted well with reduced and alkylated saposins C and A, wild-type saposin C, and saposin A [Y30A], poorly with saposin C [A31Y], and not at all with wild-type saposin A. CD spectra of wild-type and mutant saposins C and A, the corresponding neuritogenic region of saposin C, and the analogous region of saposin A showed that more "saposin C-like" molecules had neuritogenic properties. Those with more "saposin A-like" spectra did not. These studies show that the neuritogenic activity of saposin C requires specific placement of amino acids, and that Y30 of saposin A significantly alters local conformation in this critical region and suppresses neuritogenic activity.

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Anthopleurin A (ApA) and B (ApB) are 49-amino acid polypeptide toxins from the Pacific sea anemone Anthopleura xanthogrammica that interfere with inactivation of voltage-gated sodium channels. ApA, which differs from ApB in seven of the 49 amino acids, displays markedly enhanced isoform selectivity compared with ApB, acting preferentially on cardiac over neuronal sodium channels. Previous studies in this lab have indicated the importance of two unique charged residues in ApB, Arg-12 and Lys-49, in this toxin's ability to discriminate between neuronal and cardiac sodium channels. Likewise, a double mutant (R12S/K49Q) recently characterized in this lab (Khera et al., 1995) displays a greatly reduced affinity for neuronal channels, essentially restoring the discriminatory ability of ApA. When the remaining five residues unique to ApB are individually converted to those of ApA, only ApB (Pro-13) shows a major effect, reducing the affinity of the new mutant toxin (P13V) against both channel isoforms approximately 10-fold. This effect is most likely the result of a conformational rearrangement within the surrounding cationic cluster which includes Arg-12 and -14, as well as Lys-49. However, when placed into the context of the double mutant R12S/K49Q a unique effect is observed: the new triple mutant (R12S/P13V/K49Q) is no longer able to discriminate effectively between channel isoforms. Its affinity for the neuronal sodium channel is significantly enhanced compared to either P13V or to the double mutant R12S/K49Q. These results are consistent both with our proposed model (Khera et al., 1995) and with the recently reported solution structure of ApB, which implicate the cationic cluster in both affinity and channel isoform selectivity. We suggest that the P13V mutation results in a shift in the relative orientation of cationic residues within the large flexible loop between residues 9-18, thus strengthening their interactions with target sequences of the neuronal sodium channel.