Genomic and functional characterization of the diverse immunoglobulin domain-containing protein (DICP) family

A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs vary between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and affect parallel patterns of ligand recognition that potentially impact species-specific advantages.     

Trans fatty acid derived phospholipids show increased membrane cholesterol and reduced receptor activation as compared to their cis analogs

The consumption of trans fatty acid (TFA) is linked to the elevation of LDL cholesterol and is considered to be a major health risk factor for coronary heart disease. Despite several decades of extensive research on this subject, the underlying mechanism of how TFA modulates serum cholesterol levels remains elusive. In this study, we examined the molecular interaction of TFA-derived phospholipid with cholesterol and the membrane receptor rhodopsin in model membranes. Rhodopsin is a prototypical member of the G-protein coupled receptor family. It has a well-characterized structure and function and serves as a model membrane receptor in this study. Phospholipid-cholesterol affinity was quantified by measuring cholesterol partition coefficients. Phospholipid-receptor interactions were probed by measuring the level of rhodopsin activation. Our study shows that phospholipid derived from TFA had a higher membrane cholesterol affinity than their cis analogues. TFA phospholipid membranes also exhibited a higher acyl chain packing order, which was indicated by the lower acyl chain packing free volume as determined by DPH fluorescence and the higher transition temperature for rhodopsin thermal denaturation. The level of rhodopsin activation was diminished in TFA phospholipids. Since membrane cholesterol level and membrane receptors are involved in the regulation of cholesterol homeostasis, the combination of higher cholesterol content and reduced receptor activation associated with the presence of TFA-phospholipid could be factors contributing to the elevation of LDL cholesterol.     

Identification of a methylation imprint mark within the mouse Gnas locus

The imprinted mouse gene Gnas produces the G protein alpha-subunit G(S)alpha and several other gene products by using alternative promoters and first exons. G(S)alpha is maternally expressed in some tissues and biallelically expressed in most other tissues, while the gene products NESP55 and XLalphas are maternally and paternally expressed, respectively. We investigated the mechanisms of Gnas imprinting. The G(S)alpha promoter and first exon are not methylated on either allele. A further upstream region (approximately from positions -3400 to -939 relative to the G(S)alpha translational start site) is methylated only on the maternal allele in all adult somatic tissues and in early postimplantation development. Within this region lies a fourth promoter and first exon (exon 1A) that generates paternal-specific mRNAs of unknown function. Exon 1A and G(S)alpha mRNAs have similar expression patterns, making competition between their promoters unlikely. Differential methylation in this region is established during gametogenesis, being present in oocytes and absent in spermatozoa, and is maintained in preimplantation E3. 5d blastocysts. Therefore, this region is a methylation imprint mark. In contrast, differential methylation of the NESP55 and XLalphas promoter regions (Nesp and Gnasxl) is not established during gametogenesis. The methylation imprint mark that we identified may be important for the tissue-specific imprinting of G(S)alpha.     

Influence of cholesterol on equilibrium and dynamic bilayer structure of unsaturated acyl chain phosphatidylcholine vesicles as determined from higher order analysis of fluorescence anisotropy decay

The influence of cholesterol on equilibrium and dynamic bilayer structure in minimally to highly unsaturated phosphatidylcholine (PC) vesicles has been examined by characterization of the dynamic fluorescence properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Large, unilamellar egg PC, palmitoyloleoyl-PC (POPC), dioleoyl-PC (DOPC), palmitoylarachidonoyl-PC (PAPC), and palmitoyldocosahexaenoyl-PC (P-22:6-PC) vesicles containing no cholesterol or approximately 15 or 30 mol % cholesterol have been examined. Equilibrium and dynamic DPH orientational properties were analyzed according to an orthogonal, bimodal orientational distribution function [Straume, M., & Litman, B.J. (1987) Biochemistry (preceding paper in this issue)]. The same mathematical formalism was applied to TMA-DPH except that probe orientational probability was permitted only in the distribution peak aligned parallel to the bilayer normal. TMA-DPH fluorescence lifetimes were consistently increased by incorporation of cholesterol into these vesicles. Greater acyl chain unsaturation and increasing temperature each promoted reduction of lifetimes in the presence or absence of cholesterol. DPH lifetimes were much less sensitive than those of TMA-DPH to changes in composition or temperature. This behavior is consistent with reduced water penetrability into liquid-crystalline bilayers as cholesterol content is increased and as acyl chain unsaturation and temperature are reduced. Cholesterol also induces substantial equilibrium ordering of the bilayer both at the hydrophobic core and at the bilayer-water interface. DPH orientational distributions were shifted in favor of alignment parallel to the acyl side chains. The distributions of both probes were narrowed in response to incorporation of cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential perturbation of erythrocyte membrane function by structurally related polycyclic aromatic hydrocarbons

Examination of the interaction of a number of structurally related polycyclic aromatic hydrocarbons with the erythrocyte plasma membrane indicated that the presence and position of methyl groups on the lipophilic hydrocarbon nucleus determined whether the compound acted as an inhibitor of membrane function. 7,12-Dimethylbenz(a)anthracene, a potent carcinogen, acted as a noncompetitive inhibitor of membrane acetylcholinesterase. The inhibition depended on the anion composition of the buffer at the time of exposure of the cells to inhibitor, i.e., it was only manifest in the presence of an anion gradient. The temperature dependence of the intact cell enzyme in the presence of inhibitor was influenced by the temperature at which the compound was added prior to assay and may involve the perturbation of tightly associated lipids. Glucose exchange across the membrane was inhibited by the same compounds which inhibit acetylcholinesterase. The temperature dependence of the exchange was not grossly altered by the presence of 7,12-dimethylbenz(a)anthracene. The observed inhibition of two membrane functions by the polycyclic aromatic hydrocarbons does not correlate simply with their theoretical octanol/water partition coefficients, water solubilities, or ability to confer membrane stabilization against osmotic hemolysis. This demonstration of differential inhibition by compounds having the same overall hydrophobicity was unexpected and suggests a more complex mode of interaction with the cell membrane.     

Investigations of the molecular basis for the temperature-dependent insolubility of cryoglobulins. VI. Quenching by acrylamide of the intrinsic tryptophan fluorescence of cryoglobulin and non-cryoglobulin IgM proteins

The acrylamide-quenching patterns of the intrinsic tryptophan fluorescence of six cold-soluble monoclonal immunoglobulin M (IgM) and two monoclonal IgM proteins possessing cryoglobulin properties (abnormal cold insolubility) have been compared. Static and dynamic components of quenching have been resolved by a modified form of the Stern-Volmer relationship. The unusual observation of static quenching seen with the multitryptophan containing IgM is determined to be a consequence of essentially homogeneous indole fluorescence arising from conserved tryptophan residues within each homologous immunoglobulin domain. Although the static component of the quenching of the two IgM cryoimmunoglobulins examined is similar to that of the non-cryoimmunoglobulin, IgM, some of the cryoglobulin's tryptophan residues appear to be more kinetically exposed to acrylamide than the tryptophans in the non-cryoglobulin IgM. An unusually large negative entropy of activation observed for the quenching process of both cryoimmunoglobulins suggests some abnormality in the dynamic (flexibility) properties of these proteins.