Lipid mixing between lipoplexes and plasma lipoproteins is a major barrier for intravenous transfection mediated by cationic lipids

It has been previously shown that transfection activity of cationic liposome/DNA lipoplexes delivered systemically is drastically inhibited by lipoproteins (Tandia, B. M., Vandenbranden, M., Wattiez, R., Lakhdar, Z., Ruysschaert, J. M., and Elouahabi, A. (2003) Mol Ther. 8, 264-273). In this work, we have compared the binding/uptake and transfection activities of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) and diC14-amidine (3-tetradecylamino-N-tert-butyl-N'-tetra-decylpropionamidine)-containing lipoplexes in the presence or absence of purified low density lipoproteins and high density lipoprotein. Binding/uptake of both lipoplexes by the mouse lung endothelial cell line was inhibited to a similar extent in the presence of lipoproteins. In contrast, transfection activity of diC14-amidine-containing lipoplexes was almost completely inhibited (approximately by 95%), whereas approximately 40% transfection activity of DOTAP-containing lipoplexes was preserved in the presence of lipoproteins. Interestingly, the ability of lipoproteins to inhibit the transfection efficiency of lipoplexes was well correlated with their ability to undergo lipid mixing with the cationic lipid bilayer as revealed by fluorescence resonance energy transfer assay. Incubation of lipoplexes with increased doses of lipoproteins resulted in enhanced lipid mixing and reduced transfection activity of the lipoplexes in mouse lung endothelial cells. The role of lipid mixing in transfection was further demonstrated using lipid-mixing inhibitor, lyso-phosphatidylcholine, or activator (dioleoylphosphatidylethanolamine). Incorporation of Lyso-PC into diC14-amidine-containing lipoplexes completely abolished their capacity to undergo lipid mixing with lipoproteins and allowed them to reach a high transfection efficiency in the presence of lipoproteins. On the other hand, the incorporation of dioleoylphosphatidylethanolamine into DOTAP/DNA lipoplex activated lipid mixing with the lipoproteins and was shown to be detrimental toward the transfection activity of these lipoplexes. Taken together, these results indicate that fusion of lipoplexes with lipoproteins is a limiting factor for in vivo transfection.     

Proton nuclear magnetic resonance study on the dynamics of the conformation of the hinge segment of human G1 immunoglobulin

A proton nuclear magnetic resonance (NMR) study is reported for the dynamics of the conformation of the hinge segment of human G1 immunoglobulin. The hinge fragment (Thr223-His-Thr-Cys-Pro-Pro-Cys-Pro-Ala-Pro-Glu-Leu234)2 was obtained by tryptic digestion of F(ab')2, a peptic fragment of IgG1. Comparisons of the NMR results obtained for the hinge fragment with those for the intact IgG1 and its fragments led us to conclude that a significant change in conformation of the segment preceding the disulfide-linked Cys-Pro-Pro-Cys core is induced when the Fab portion is cleaved off and the presence or absence of the Fc portion affects very little, if any, of the conformation of this part of the hinge. On the basis of the present NMR results along with those which we have obtained previously using the intact IgG1 and its fragments, it was concluded that the conformation of the segment preceding the Cys-Pro-Pro-Cys core of the intact IgG1 can be maintained only when it is flanked by the Fab portion and the Cys-Pro-Pro-Cys core. An X-ray crystallographic study [Marquart, M., Deisenhofer, J., Huber, R., & Palm, W. (1980) J. Mol. Biol. 141, 369-392] showed that segment Cys-220-Thr-225 forms a one-turn helix with little inherent stability. Upon loss of Fab or Fc, residual segments of the hinge would become too short to form the helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coatomer-bound Cdc42 regulates dynein recruitment to COPI vesicles

Cytoskeletal dynamics at the Golgi apparatus are regulated in part through a binding interaction between the Golgi-vesicle coat protein, coatomer, and the regulatory GTP-binding protein Cdc42 (Wu, W.J., J.W. Erickson, R. Lin, and R.A. Cerione. 2000. Nature. 405:800-804; Fucini, R.V., J.L. Chen, C. Sharma, M.M. Kessels, and M. Stamnes. 2002. Mol. Biol. Cell. 13:621-631). The precise role of this complex has not been determined. We have analyzed the protein composition of Golgi-derived coat protomer I (COPI)-coated vesicles after activating or inhibiting signaling through coatomer-bound Cdc42. We show that Cdc42 has profound effects on the recruitment of dynein to COPI vesicles. Cdc42, when bound to coatomer, inhibits dynein binding to COPI vesicles whereas preventing the coatomer-Cdc42 interaction stimulates dynein binding. Dynein recruitment was found to involve actin dynamics and dynactin. Reclustering of nocodazole-dispersed Golgi stacks and microtubule/dynein-dependent ER-to-Golgi transport are both sensitive to disrupting Cdc42 mediated signaling. By contrast, dynein-independent transport to the Golgi complex is insensitive to mutant Cdc42. We propose a model for how proper temporal regulation of motor-based vesicle translocation could be coupled to the completion of vesicle formation.     

The crystallizable human myeloma protein Dob has a hinge-region deletion.

During experiments to prepare heavy-metal derivatives of the crystallizable human IgG1 (k) immunoglobulin Dob, it became apparent that this protein has several unusual features. (1) Instead of the four labile interchain disulfide bridges ordinarily found in IgG1, the Dob protein has only a single interchain disulfide bridge, which connects its two light chains. (2) The Dob heavy chain appears to be slightly smaller than a control gamma1 chain, as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by gel filtration in guanidine. (3) The Dob heavy chain has three fewer residues of half-cystine than expected in gamma1 chains. (4) The Dob IgG is relatively resistant to digestion with papain and trypsin; however, it is readily digested with pepsin, although at an unusual site. These findings suggest that some or all of the gamma1 hinge region is missing in Dob. To localize the deletion, we prepared an F(ab')2 fragment consisting of two heavy-chain pieces (Fd') noncovalently associated with the light-chain dimer. The Fd' piece was isolated and digested with trypsin. The sequence of the C-terminal tryptic peptide was Val-Ala-Pro-Glu-Leu-Leu-Gly-Gly-Pro-Ser-Val. Positions 2-11 of this peptide are identical with residue positions 231-240 of the gamma1 chain. The N-terminal valine could be either Val-211 or Val-215 of the gamma1 sequence. A tryptic peptide, Val-Asp-Lys-Lys, was also isolated from Dob Fd'; this sequence is not found in the variable region of the Dob heavy chain [Steiner, L. A., Garcia Pardo, A., & Margolies, M. N. (1979) Biochemistry (following paper in this issue)] but corresponds to positions 211-214 of the gamma1 constant region. Therefore, the deletion cannot include these residues and must begin after Val-215; normal gamma1 sequence resumes at Ala-231. The same 15-residue deletion has been found in two other IgG1 proteins, Mcg [Fett, J. W., Deutsch, H. F., & Smithies, O. (1973) Immunochemistry 10, 115] and Lec [Rivat, C., Schiff, C., Rivat, L., Ropartz, C., & Fougereau, M. (1976) Eur. J. Immunol. 6, 545]. Possible explanations for the occurrence of identical hinge-region deletions in three different immunoglobulins are suggested by recent experiments demonstrating that the three constant domains and the hinge region of mouse gamma1 chains are each encoded by separate segments of DNA [Sakano, H., Rogers, J. H., Hüppi, K., Brack, C., Traunecker, A., Maki, R., Wall, R., & Tonegawa, S. (1979) Nature (London) 277, 627].     

The influence of lipid composition on glycophorin-induced bilayer permeability

Glycophorin was incorporated into large unilamellar vesicles and the bilayer permeability was measured as a function of the lipid composition. In agreement with previous data (Van der Steen, A.T.M., De Kruijff, B. and De Gier, J. (1982) Biochim. Biophys. Acta 691, 13-23) it was found that glycophorin greatly increased the bilayer permeability of DOPC vesicles. This effect was observed for a large variety of phosphatidylcholines, differing in their fatty acid composition and homogeneity. In sharp contrast, it was observed that variations in the polar headgroups by incorporation of DOPE, DOPS and, to a lesser extent, cholesterol, into the DOPC/glycophorin vesicles restored the barrier function. These results are compared to the size of the particles, revealed by freeze-fracture electron microscopy on the glycophorin-containing bilayer and are discussed in the light of various types of lipid-protein interactions and protein aggregation state.     

Mechanism of inhibition of mitochondrial enzymatic complex I-III by adriamycin derivatives

We demonstrate here that complex I-III of bovine heart mitochondrial membrane is inhibited by adriamycin derivatives. This inhibition is a cardiolipin-dependent process. This lipid, specific to the inner mitochondrial membrane, has been shown previously to interact specifically with adriamycin in model membranes (Goormaghtigh, E., Chatelain, P., Caspers, J. and Ruysschaert, J.-M. (1980) Biochim. Biophys. Acta 597, 1-14) and in mitochondrial membranes (Cheneval, D., Müller, M., Toni, R., Ruetz, S. and Carafoli, E. (1985) J. Biol. Chem. 260, 13003-13007). The differential scanning calorimetry data indicate that, in multilamellar liposomes, the formation of antibiotic-cardiolipin complexes induces a clustering of cardiolipin molecules. Conformational analysis of the antibiotic-cardiolipin complexes suggests that plane-plane interactions between the antibiotics aromatic moieties stabilize this complex formation. Possible mechanisms of inactivation of complex I-III by adriamycin are proposed.     

A mismatch between the length of gramicidin and the lipid acyl chains is a prerequisite for HII phase formation in phosphatidylcholine model membranes

Previously it was shown that gramicidin can induce HII phase formation in diacylphosphatidylcholine model membranes only when the lipid acyl chain length exceeds 16 carbon atoms (Van Echteld, C.J.A., De Kruijff, B., Verkleij, A.J., Leunissen-Bijvelt, J. and De Gier, J. (1982) Biochim. Biophys. Acta 692, 126-138). Using 31P-NMR and small angle X-ray diffraction we now demonstrate that upon increasing the length of gramicidin, the peptide loses its ability to induce HII phase formation in di-C18:1c-PC but not in the longer chained di-C22:1c-PC. It is concluded that a mismatch in length between gramicidin and the lipid acyl chains, when the latter would provide excess bilayer thickness, is a prerequisite for HII phase formation in phosphatidylcholine model membranes.     

Structural and functional characterization of disulfide isoforms of the human IgG2 subclass

In the accompanying report ( Wypych, J., Li, M., Guo, A., Zhang, Z., Martinez, T., Allen, M. J., Fodor, S., Kelner, D. N., Flynn, G. C., Liu, Y. D., Bondarenko, P. V., Ricci, M. S., Dillon, T. M., and Balland, A. (2008) J. Biol. Chem. 283, 16194-16205 ), we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1beta ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 m guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 > IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2lambda composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "blood-like" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.     

Saposin A mobilizes lipids from low cholesterol and high bis(monoacylglycerol)phosphate-containing membranes: patient variant Saposin A lacks lipid extraction capacity

Saposin A (Sap-A) is one of five known sphingolipid activator proteins required for the lysosomal degradation of sphingolipids and for the loading of lipid antigens onto antigen-presenting molecules of the CD1 type. Sap-A assists in the degradation of galactosylceramide by galactosylceramide-beta-galactosidase in vivo, which takes place at the surface of intraendosomal/intralysosomal vesicles. Sap-A is believed to mediate the interaction between the enzyme and its membrane-bound substrate. Its dysfunction causes a variant form of Krabbe disease. In the present study we prepared glycosylated Sap-A free of other Saps, taking advantage of the Pichia pastoris expression system. Using liposomes and surface plasmon resonance spectroscopy, we tested the binding and lipid mobilization capacity of Sap-A under different conditions. Along the endocytic pathway, the pH value decreases, and the lipid composition of intraendosomal and intralysosomal membranes changes drastically. In the inner membranes the cholesterol concentration decreases, and that of the anionic phospholipid bis(monoacylglycero)phosphate increases. Here, we show that Sap-A is able to bind to liposomes and to mobilize lipids out of them at acidic pH values below pH 4.7. Low cholesterol levels and increasing concentrations of bis(monoacylglycero)phosphate favor lipid extraction significantly. Galactosylceramide as a bilayer component is not essential for lipid mobilization by Sap-A, which requires intact disulfide bridges for activity. We also show for the first time that glycosylation of Sap-A is essential for its lipid extraction activity. Variant Sap-A proteins, which cause storage of galactosylceramide in humans (Krabbe disease, Spiegel, R., Bach, G., Sury, V., Mengistu, G., Meidan, B., Shalev, S., Shneor, Y., Mandel, H., and Zeigler, M. (2005) Mol. Genet. Metab. 84, 160-166) and in mutant mice (Matsuda, J., Vanier, M. T., Saito, Y., Tohyama, J., and Suzuki, K. (2001) Hum. Mol. Genet. 10, 1191-1199) are deficient in lipid extraction capacity.     

Approach to the direct intramolecular localization of antigenic determinants in Androctonus australis hemocyanin with monoclonal antibodies by molecular immunoelectron microscopy

Monoclonal antibodies (mAb) directed vs. subunits from hemocyanin (Hc) of the scorpion Androctonus australis were used in molecular immunoelectron microscopy (MIEM) to directly localize the epitopes within the subunits. Four types of mAb were used. First, mAb 6302, an IgG clone highly specific for subunit Aa 2, produced with native hemocyanin long strings composed of hemocyanin molecules in the side view and in the 45 degrees view. At lower concentration, "parachute" and "butterfly" structures composed of two Hc molecules and one monoclonal immunoglobin G (IgG) molecule were obtained. Fab fragments prepared from mAb 6302 bound exactly on the top and bottom edges of the molecule. The second type of mAb (6003), directed vs. subunit Aa 2, produced nice immunocomplexes with the free subunit but nothing with the native oligomer. It is suggested that due to steric hindrance or to conformational changes the epitope is not accessible in the native molecule. The third mAb belonged to the IgM class and apparently bound Hc in the Aa 2 area. However, because of the difficulty of separating the immunocomplexes from the residual mAb and the polymorphism of the IgM molecules, monoclonal IgM are no longer used for MIEM. The last type of mAb (5701) had a high affinity and a high specificity for subunit Aa 6. It produced two types of immunocomplexes with native Hc. The two types differed by a 180 degrees rotation around one of the Fab arms. These complexes, which support recent results of Wrigley et al. [Wrigley, N. G., Brown, E. B., & Skehel, J. J. (1983) J. Mol. Biol. 169, 771-774] and of Roux [Roux, K. H. (1983) Eur. J. Immunol. 14, 459-464], indicate that monoclonal IgG have a high degree of rotational flexibility around the Fab arm. Monoclonal antibody 5701 bound exactly at the corner of the molecule in the area where subunit Aa 6 is known to be located. The MIEM approach of the location of the epitope requires the model of the architecture and of the quaternary structure to be very precise. Thus, recent findings of Gaykema et al. [Gaykema, W. P. J., Hol, J. M., Vereijken, J. M., Soeter, N. M., Bak, H. J., & Beintema, J. J. (1984) Nature (London) 309, 23-29] and of Van Heel et al. [Van Heel, M., Keegstra, W., Schutter, W., & Van Bruggen, E. F. J. (1983) Life Chem. Rep., Suppl. Ser. 1, 69-73] led to a reexamination of previous models.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diffusion of dihydropyridine calcium channel antagonists in cardiac sarcolemmal lipid multibilayers.

A membrane bilayer pathway model has been proposed for the interaction of dihydropyridine (DHP) calcium channel antagonists with receptors in cardiac sarcolemma (Rhodes, D.G., J.G. Sarmiento, and L.G. Herbette. 1985. Mol. Pharmacol. 27:612-623) involving drug partition into the bilayer with subsequent receptor binding mediated (though probably not rate-limited) by diffusion within the bilayer. Recently, we have characterized the partition step, demonstrating that DHPs reside, on a time-average basis, near the bilayer hydrocarbon core/water interface. Drug distribution about this interface may define a plane of local concentration for lateral diffusion within the membrane. The studies presented herein examine the diffusional dynamics of an active rhodamine-labeled DHP and a fluorescent phospholipid analogue (DiIC16) in pure cardiac sarcolemmal lipid multibilayer preparations as a function of bilayer hydration. At maximal bilayer hydration, the drug diffuses over macroscopic distances within the bilayer at a rate identical to that of DiI (D = 3.8 X 10(-8) cm2/s), demonstrating the overall feasibility of the membrane diffusion model. The diffusion coefficients for both drug and lipid decreased substantially as the bilayers were dehydrated. While identical at maximal hydration, drug diffusion was significantly slower than that of DiIC16 in partially dehydrated bilayers, probably reflecting differences in mass distribution of these probes in the bilayer.     

Interaction of leukocyte integrins with ligand is necessary but not sufficient for function

The leukocyte integrins (CD11/CD18 or beta 2-type integrins) are expressed exclusively on leukocytes and participate in many adhesion- dependent functions (Arnaout, M.A. 1990. Blood. 75:1037-1050; Springer, T. A. 1990. Nature. (Lond.) 346:425-434; Dustin, M. L., and T. S. Springer. 1991. Annu. Rev. Immunol. 9:27-66). The avidity of leukocyte integrin binding to their ligands or counter-receptors is dependent upon response to intracellular signals (Wright, S. D., and B. C. Meyer. 1986. J. Immunol. 136:1759-1764; Dustin, M. A., and T. S. Springer. 1989. Nature (Lond.). 341:619-624). We have investigated the effects of a novel mAb (mAb 24) which defines a leukocyte integrin alpha subunit epitope that is Mg(2+)-dependent and may be used as a "reporter" of the activation state of these receptors (Dransfield, I., and N. Hogg. 1989. EMBO (Eur. Mol. Biol. Organ) J. 8:3759-3765; Dransfield, I., A.-M. Buckle, and N. Hogg. 1990. Immunol. Rev. 114:29-44; Dransfield, I., C. Cabanas, A. Craig, and N. Hogg. 1992. J. Cell Biol.) Data is presented to show that this mAb inhibits monocyte-dependent, antigen-specific T cell proliferation and IL-2-activated natural killer cell assays which are both dependent on lymphocyte function-associated antigen-1 (LFA-1), and complement receptor type 3 (CR3)-mediated neutrophil chemotaxis to f-Met-Leu-Phe. This inhibitory effect is not caused by the prevention of receptor/ligand binding because LFA-1/ICAM-1, LFA-1/ICAM-2,3 and CR3/iC3b interactions are, under activating conditions, promoted rather than blocked by mAb 24. As it does not interfere with mitogen- stimulated T cell proliferation, it is unlikely that mAb 24 transduces a "negative" or antiproliferative signal to the T cells to which it is bound. Using a model system of transient activation of LFA-1, we have found that mAb 24 prevents "deadhesion" of receptor/ligand pairs, possibly locking leukocyte integrins in an "active" conformation. It is speculated that inhibition of leukocyte integrin function by this mAb reflects the necessity for dynamic leukocyte integrin/ligand interactions.     

Structure of a mouse immunoglobulin G that lacks the entire CH1 domain: protein sequencing and small-angle X-ray scattering studies

The structure of a short-chain IgG2a antibody, which is a member of the family of mouse anti-dansyl switch variant antibodies with identical variable regions but different heavy-chain constant regions [Dangl, J.L., Parks, D. R., Oi, V. T., & Herzenberg, L. A. (1982) Cytometry 2, 395-401], is reported. Amino acid sequencing analyses have demonstrated that in the short-chain IgG2a antibody the entire CH1 domain is deleted whereas the hinge region remains intact. Small-angle X-ray scattering data were collected for the short-chain IgG2a antibody and compared with those for the switch variant IgG1, IgG2a, and IgG2b antibodies with the normal heavy chain. It has been concluded that deletion of the CH1 domain results in a large structural change and the short-chain IgG2a antibody possesses an elongated molecular shape with a much smaller hinge angle as compared with the normal IgG2a antibody that is a Y-shaped molecule.     

Interaction of C-terminal loop 13 of sodium-glucose cotransporter SGLT1 with lipid bilayers

We have previously shown that C-terminal loop 13 of SGLT1 acts as a major binding domain for the aglucon residues of d-glucose transport inhibitors, phlorizin (Raja, M. M., Tyagi, N. K., and Kinne, R. K. H. (2003) Phlorizin Recognition in a C-terminal Fragment of SGLT1 Studied by Tryptophan Scanning and Affinity Labeling, J. Biol. Chem. 278, 49154-49163) and alkyl glucosides (Raja, M. M., Kipp, H., and Kinne, R. K. H. (2004) C-Terminus Loop 13 of Na(+) Glucose Cotransporter SGLT1 Contains a Binding Site for Alkyl Glucosides, Biochemistry 43, 10944-10951). Topology of this loop with regard to the membrane lipids is hitherto a point of debate. Here we report on in vitro incorporation studies using fluorescence of Trp mutants of loop 13 to determine the position of various parts of the loop with the lipid bilayer. Six single Trp mutants were prepared as described in previous studies (Raja et al., 2003) and subsequently incorporated into DOPC:DOPG (60:40% molar ratio) lipid vesicles. Upon addition of the phospholipids only one mutant, R601W, exhibited no change in the fluorescence intensities, position of maxima, or acrylamide accessibility. Mutants Q581W, E621W, and L630W exhibited the most pronounced blue shifts (3-6 nm) and protection against acrylamide, suggesting a position of these segments within the lipid bilayer. This assumption was confirmed by the result that the fluorescence of only these mutants was quenched by doxyl spin membrane embedded labels in the 5- or 12-positions of the acyl side chain of phospholipids. The other parts of the peptide appear to remain outside of the lipid vesicles. Trp-591 and Trp-611 showed, although to a different extent, increase in fluorescence, blue shift of maxima, and decrease in acrylamide accessibility but no interaction with the spin-labeled phospholipids. This suggests changes in the conformation of the peptide itself. These conformation changes are probably induced by the interaction of an adjacent lysine rich region of the peptide with the negatively charged DOPG, since in the absence of this lipid no incorporation of loop 13 into the bilayer is observed. Trypsin cleavage experiments of loop 13 in proteoliposomes yield a peptide containing amino acid residues 603 to 614, confirming that this part of the loop is accessible at the extravesicular face of the membranes. The studies show that at least in the in vitro system the part of loop 13 essential for the interaction with the transport inhibitors is located extracellularly, making a similar arrangement in the intact SGLT1 probable.     

Biochemical characterization of the phospholipase A2 purified from the venom of the Mexican beaded lizard (Heloderma horridum horridum Wiegmann)

A phospholipase A2 was isolated from the venom of the mexican beaded lizard (Heloderma horridum horridum) by phenyl-Sepharose chromatography followed by Sephadex G-75 gel filtration and two additional steps on ion exchange resins (DE-32 cellulose). The affinity chromatographic method (PC-Sepharose 4B) reported for the isolation of other phospholipases [Rock, Ch. O., & Snyder, F. (1975) J. Biol. Chem. 250, 2564-2566; King, T. P., Alagon, A. C., Kwan, J., Sobotka, A. K., & Lichteinstein, L. M. (1983) Mol. Immunol. 20, 297-308; King, T. P., Kochoumian, L., & Joslyn, A. (1984) Arch. Biochem. Biophys. 230, 1-12] was uneffective for the separation of this enzyme. The monomeric form of the Heloderma phospholipase has an apparent Mr of 18 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 19 060 as calculated from amino acid analysis. It also contains on the order of 7% carbohydrates per mole of enzyme. The N-terminal amino acid sequence was shown to be very different from that of phospholipases isolated from mammalian pancreas and crotalids and elapids snake venoms. The first 39 amino acid residues at the N-terminal region have 56% homology with bee venom phospholipase but differ from the bee phospholipase in that its isoelectric point is acidic (pI = 4.5), instead of basic, and it has approximately 50 amino acid residues more in the molecule. The specificity of the enzyme is mainly A2 type with possible residual B-type activity. The enzymatic activity is Ca2+-dependent. Half-cystine alignment of the Heloderma phospholipase sequence with those of other known phospholipases shows the lack of an octadecapeptide at the N-terminal region, the existence of an extra hexapeptide at positions 42-47, and an exact correspondence of Heloderma Gly-12, Gly-14, His-36, and Asp-37 with Gly-30, Gly-32, His-48, and Asp-49 from other phospholipases shown to be important for Ca2+ binding (( Dijkstra, B. W., Drenth, J., Kalk, K. H., & Vandermaalen, P. J. (1978) J. Mol. Biol. 124, 53-60 )).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and properties of type I-signal peptidases of Bacillus amyloliquefaciens.

The use of Bacillus amyloliquefaciens for enzyme production and its exceptional high protein export capacity initiated this study where the presence and function of multiple type I signal peptidase isoforms was investigated. In addition to type I signal peptidases SipS(ba) [Meijer, W.J.J., de Jong, A., Bea, G., Wisman, A., Tjalsma, H., Venema, G., Bron, S. & van Dijl, J.M. (1995) Mol. Microbiol. 17, 621-631] and SipT(ba) [Hoang, V. & Hofemeister, J. (1995) Biochim. Biophys. Acta 1269, 64-68] which were previously identified, here we present evidence for two other Sip-like genes in B. amyloliquefaciens. Same map positions as well as sequence motifs verified that these genes encode homologues of Bacillus subtilis SipV and SipW. SipU-encoding DNA was not found in B. amyloliquefaciens. SipW-encoding DNA was also found for other Bacillus strains representing different phylogenetic groups, but not for Bacillus stearothermophilus and Thermoactinomyces vulgaris. The absence of these genes, however, could have been overlooked due to sequence diversity. Sequence alignments of 23 known Sip-like proteins from Bacillus origin indicated further branching of the P-group signal peptidases into clusters represented by B. subtilis SipV, SipS-SipT-SipU and B. anthracis Sip3-Sip5 proteins, respectively. Each B. amyloliquefaciens sip(ba) gene was expressed in an Escherichia coli LepBts mutant and tested for genetic complementation of the temperature sensitive (TS) phenotype as well as pre-OmpA processing. Although SipS(ba) as well as SipT(ba) efficiently restored processing of pre-OmpA in E. coli, only SipS(ba) supported growth at TS conditions, indicating functional diversity. Changed properties of the sip(ba) gene disruption mutants, including cell autolysis, motility, sporulation, and nuclease activities, seemed to correlate with specificities and/or localization of B. amyloliquefaciens SipS, SipT and SipV isoforms.     

Role of sn-1-saturated,sn-2-polyunsaturated phospholipids in control of membrane receptor conformational equilibrium: effects of cholesterol and acyl chain unsaturation on the metarhodopsin I in equilibrium with metarhodopsin II equilibrium.

The effect of phospholipid bilayer acyl chain packing free volume on the equilibrium concentration of the form of photolyzed rhodopsin which initiates visual signal transduction, metarhodopsin II (meta II), is examined in reconstituted systems formed from the saturated phospholipid dimyristoylphosphatidylcholine (DMPC) and in the polyunsaturated phospholipid sn-1-palmitoyl-sn-2-arachidonoylphosphatidylcholine (PAPC) with and without 30 mol% cholesterol. The extent of meta II formation is determined from both flash photolysis measurements and rapidly acquired absorbance spectra. Equilibrium and dynamic properties of the lipid bilayer are characterized by the dynamic fluorescence properties of 1,6-diphenyl-1,3,5-hexatriene (DPH). DPH orientational properties are characterized by fv, a parameter which reflects the volume available for probe reorientation in the bilayer, relative to that available in an unhindered, isotropic environment [Straume, M., & Litman, B. J. (1987) Biochemistry 26, 5121-5126]. The metarhodopsin I in equilibrium with meta II equilibrium constant, Keq has a linear relationship with fv for rhodopsin in PAPC vesicles with and without cholesterol as well as for rhodopsin in DMPC vesicles, and these two correlation lines have different slopes. The correlations between Keq and fv in PAPC and DMPC systems are compared with a similar correlation in the native rod outer segment disk membrane and one reported previously in an egg phosphatidylcholine (egg PC) system [Mitchell, D. C., Straume, M., Miller, J. L., & Litman, B. J. (1990) Biochemistry 29, 9143-9149].(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipid fatty acyl chain asymmetry in the membrane bilayer of isolated skeletal muscle sarcoplasmic reticulum

We previously showed [Herbette, L. G., Blasie, J. K., DeFoor, P., Fleischer, S., Bick, R. J., Van Winkle, W. B., Tate, C. A., & Entman, M. L. (1984) Arch. Biochem. Biophys. 234, 235-242; Herbette, L. G., DeFoor, P., Fleischer, S., Pascolini, D., Scarpa, A., & Blasie, J. K. (1985) Biochim. Biophys. Acta 817, 103-122] that the phospholipid head-group distribution in the membrane bilayer of isolated sarcoplasmic reticulum is asymmetric. From these studies, both the total number of phospholipid head groups and the total lipid, as well as the head-group species for these lipids, were found to be different for each monolayer of the membrane bilayer. In this paper, we demonstrate for the first time that there is significant asymmetry in the distribution of unsaturated fatty acids between the two monolayers; i.e., the outer monolayer of the sarcoplasmic reticulum contained more unsaturated and polyunsaturated chains when compared to the inner monolayer. X-ray diffraction measurements demonstrated that the time-averaged fatty acyl chain extension for the outer monolayer was approximately 20% less than for the inner monolayer. This is consistent with the concept that the greater degree of unsaturation in the outer monolayer may provide for a decreased average fatty acyl chain extension for that layer. This architecture for the bilayer may be related to both the "resting" state mass distribution of the calcium pump protein within the membrane bilayer and possible "conformational" states of the calcium pump protein during calcium transport by the sarcoplasmic reticulum.     

Modeling the antigen combining site of an anti-dinitrophenyl antibody, ANO2.

A model structure has been constructed for a monoclonal anti-dinitrophenyl antibody. The antibody, ANO2, has been sequenced and cloned (Anglister, J., Frey, T., & McConnell, H.M., 1984, Biochemistry 23, 1138-1142). Its amino acid sequence shows striking homology with the anti-lysozyme Fab fragments HyHel5 (83%) and HyHel10 (73%). Based on this homology, a model for the ANO2 variable heavy and variable light chain framework was constructed using a hybrid of the HyHel5 light chain and the HyHel10 heavy chain backbone, omitting the hypervariable loops. These coordinates were used as scaffolds for the model building of ANO2. The CONGEN conformational sampling algorithm (Bruccoleri, R.E. & Karplus, M., 1987, Biopolymers 26, 127-196) was used to model the six hypervariable loops that contain the antigen-combining site. All the possible conformations of the loop backbones were constructed and the best loop structures were selected using a combination of the CHARMM potential energy function and evaluation of the solvent-accessible surface area of the conformers. The order in which the loops were searched was carried out based on the relative locations of the loops with reference to the framework of the beta-barrel, namely, L2-H1-L3-H2-H3-L1. The model structures thus obtained were compared to the high resolution X-ray structure (Brünger, A.T., Leahy, D.J., Hynes, T.R., & Fox, R.O., 1991, J. Mol. Biol. 221, 239-256).