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Biomedical subjects

D M Lublin

Publications and source records attributed to D M Lublin.

At least 19 recordsLinked to original sources
PubMed1997

Survival of FimH-expressing enterobacteria in macrophages relies on glycolipid traffic.

Strains of Escherichia coli persist within the human gut as normal commensals, but are frequent pathogens and can cause recurrent infection. Here we show that, in contrast to E. coli subjected to opsonic interactions stimulated by the host's immune response, E. coli that bind to the macrophage surface exclusively through the bacterial lectin FimH can survive inside the cell following phagocytosis. This viability is largely due to the attenuation of intracellular free-radical release and of phagosome acidification during FimH-mediated internalization, both of which are triggered by antibody-mediated internalization. This different processing of non-opsonized bacteria is supported by morphological evidence of tight-fitting phagosomes compared with looser, antibody-mediated phagosomes. We propose that non-opsonized FimH-expressing E. coli co-opt internalization of lipid-rich microdomains following binding to the FimH receptor, the glycosylphosphatidylinositol-linked protein CD48, because (1) the sterol-binding agents filipin, nystatin and methyl beta-cyclodextrin specifically block FimH-mediated internalization; (2) CD48 and the protein caveolin both accumulate on macrophage membranes surrounding bacteria; and (3) antibodies against CD48 inhibit FimH-mediated internalization. Our findings bring the traditionally extracellular E. coli into the realm of opportunistic intracellular parasitism and suggest how opportunistic infections with FimH-expressing enterobacteria could occur in a setting deprived of opsonizing antibodies.

Adhesins, Bacterial↗
PubMed1997

Membrane cofactor protein (CD46) is a basolateral protein that is not endocytosed. Importance of the tetrapeptide FTSL at the carboxyl terminus.

Membrane cofactor protein (MCP) is a widely distributed complement regulatory protein that is expressed on the basolateral surface of polarized epithelial cells. The basolateral targeting of the BC1 isoform of MCP was analyzed by generating deletion mutants and point mutants within the cytoplasmic tail of 16 amino acids. A sequence of four amino acids, FTSL, was found to be indispensable for the basolateral transport of MCP. This tetrapeptide has two unique features compared with the targeting motifs of other basolateral proteins: (i) it contains a phenylalanine rather than a tyrosine at position 1; (ii) it is located at the very COOH-terminal end. Replacement of the phenylalanine or the leucine by an alanine resulted in a nonpolarized delivery to the cell surface. On the other hand, substitution of a tyrosine for the phenylalanine did not affect the basolateral transport of MCP. The latter mutant, however, was efficiently internalized, whereas the wild type protein was not subject to endocytosis. Our results indicate that the targeting signal YXX-large aliphatic that is involved in various sorting events has been modulated in MCP in such a way that it allows basolateral transport but not endocytosis.

Amino Acid Sequence↗
PubMed1996

Identification of a heparin binding domain in the seventh short consensus repeat of complement factor H.

Surface polyanions such as sialic acid and heparin are thought to enhance the binding of complement factor H (fH) to C3b deposited on particles and cell surfaces, thereby reducing complement activation. fH contains 20 short consensus repeat (SCR) domains, and it has been proposed that SCR 13 contains a heparin binding site. We used recombinant proteins to determine the heparin binding site on fH. Full-length fH (H20) and truncated and SCR deletion mutant proteins were cloned and expressed in Chinese hamster ovary cells. Supernatants were applied to heparin-agarose affinity columns to determine their binding and elution profiles. Deletion of SCR 13 from H20 did not prevent heparin binding nor alter its salt elution profile, indicating that SCR 13 does not contain an essential heparin binding site. We found that SCR 7 contains a heparin binding site, as SCRs 1 through 7 were the smallest truncated proteins to bind heparin (89 +/- 3%). Furthermore, deletion of SCR 7 from a protein containing SCRs 1 through 9 reduced heparin binding, whereas deletion of SCR 6 did not (17 +/- 13 vs 81 +/- 13%; p = 0.02). It is likely that other heparin binding sites exist within SCRs 10 through 20; an SCR 7 deletion mutant of H20 eluted earlier than H20, but still showed >99% binding to immobilized heparin. SCR 13 does not contain such a site because a double deletion of SCRs 7 and 13 from H20 showed >97% heparin binding and had an elution profile smilar to that of a single deletion of SCR 7.

Animals↗
PubMed1996

CD36 is palmitoylated on both N- and C-terminal cytoplasmic tails.

The membrane protein CD36 has been reported to carry out a wide range of potential functions, including serving as a receptor for thrombospondin, collagen, oxidized low density lipoprotein, fatty acids, anionic phospholipids, and Plasmodium falciparum malaria parasitized erythrocytes. This implicates CD36 in cellular adhesion, human atherosclerotic lesion formation, lipid metabolism, and malaria. A presumed rat homolog of CD36 was previously reported to be palmitoylated. We confirmed that human CD36 is palmitoylated and identified cysteines 3, 7, 464, and 466 as the palmitoylation sites using a mutagenesis approach. This result suggests that both the N- and C-terminal tails of CD36 are cytoplasmic. Published models for the topology of CD36 have the C terminus located in the cytoplasm but differ as to whether the N terminus is cytoplasmic or extracellular. To address this question, a C-terminal truncation mutant of CD36 was made by introducing a stop codon just upstream of the C-terminal transmembrane domain. This mutant was found membrane-bound when expressed in human embryonic kidney 293 cells, indicating that the N-terminal hydrophobic domain serves as a transmembrane anchor, and thus supporting a CD36 topology with two transmembrane domains.

Animals↗
PubMed1996

Activation of the gene encoding decay accelerating factor following nerve growth factor treatment of sensory neurons is mediated by promoter sequences within 206 bases of the transcriptional start site.

Using two independent differential screening procedures designed to identify novel mRNAs induced by nerve growth factor (NGF) treatment of adult dorsal root ganglion (DRG) neurons, we have isolated cDNA clones derived from the gene encoding decay accelerating factor (DAF). Hybridization analysis and semi-quantitative polymerase chain reaction confirmed that the DAF mRNA was indeed induced in NGF-treated adult DRG neurons. Moreover, the DAF gene promoter is NGF inducible (approximately two- to threefold) when transfected into DRG neurons, and this effect is primarily dependent on sequences between -206 and -77 relative to the transcriptional start site. Hence, the DAF gene constitutes a novel NGF-inducible gene whose mRNA is elevated in response to NGF treatment of DRG neurons. The potential significance of this effect is discussed in terms of the role of NGF in modulating the transcriptional activity and function of adult DRG neurons.

Animals↗
PubMed1996

Oligomerization of VIP21-caveolin in vitro is stabilized by long chain fatty acylation or cholesterol.

VIP21-caveolin is one of the components which form the cytoplasmic surface of caveolae. In vivo, this integral membrane protein is found in homo-oligomers with molecular masses of approximately 200, 400 and 600 kDa. These oligomers are also formed by the addition of cytosol to the in vitro synthesized and membrane inserted VIP21-caveolin. Here we show that long chain fatty acyl coenzyme A esters can completely substitute for cytosol in inducing 200 kDa and 400 kDa complexes, whereas 25-hydroxy-cholesterol can produce the 200 kDa oligomer. In order to understand whether acylation of VIP21-caveolin itself is a prerequisite for oligomerization, we studied a mutant protein lacking all three cysteines. When analyzed by velocity sucrose gradient centrifugation in the presence of the non-ionic detergent octylglucoside, both palmitoylated and non-palmitoylated VIP21-caveolin formed oligomers that were indistinguishable. However, only the oligomers of the non-palmitoylated protein are disrupted when analyzed by SDS-PAGE without boiling. These data suggest that the protein domains of VIP21-caveolin are the primary determinants of oligomerization, but that palmitoylation of cysteine residues can increase the stability of the oligomers.

Acyl Coenzyme A↗
PubMed1996

Expression of functional decay-accelerating factor (CD55) in transgenic mice protects against human complement-mediated attack.

Transgenic mice expressing human CD55 were generated by microinjection of a CD55-minigene under the control of the mouse H2K(b) (MHC class I) promoter. Offspring were tested for transgene integration by PCR analysis, and for CD55 expression on peripheral blood leukocytes (PBLs) by flow cytometry. Expression levels of 15 founders ranged from 30 to 80% of that on human neutrophils. Immunohistochemical analysis of kidney, heart, liver, and lung tissue demonstrated staining for CD55 on endothelial surfaces as well as general diffuse staining throughout the tissues. The capacity of the transgenically expressed CD55 to prevent human C3 deposition on the surface of mouse splenocytes was assessed by flow cytometry. Cells from hemizygous mice incubated with 10% fresh human serum as a source of natural antibody and complement bound approximately 65% less C3 than control littermates. No further protection was seen using cells from homozygous littermates, and the protective effect was abrogated by prior incubation with an OFFi-CD55 monoclonal antibody. Similarly, transgenic mice were afforded significant protection from human serum-mediated lysis, determined using an LDH release assay. Hearts perfused with human plasma showed no increase in survival time in a modified Langendorff perfusion system, however deposition of human C3c was greatly reduced in transgenic hearts.

Animals↗
PubMed1996

Control of the complement system.

The complement system has developed a remarkably simple but elegant manner of regulating itself. It has faced and successfully dealt with how to facilitate activation on a microbe while preventing the same on host tissue. It solved this problem primarily by creating a series of secreted and membrane-regulatory proteins that prevent two highly undesirable events: activation in the fluid phase (no target) and on host tissue (inappropriate target). Also, if not checked, even on an appropriate target, the system would go to exhaustion and have nothing left for the next microbe. Therefore, the complement enzymes have an intrinsic instability and the fluid-phase control proteins play a major role in limiting activation in time. The symmetry of the regulatory process between fluid phase and membrane inhibitors at the C4/C3 step of amplification and convertase formation as well as at the MAC steps are particularly striking features of the self/nonself discrimination system. The use of glycolipid anchored proteins on membranes to decay enzymes and block membrane insertion events is unlikely to be by chance. Finally, it is economical for the cofactor regulatory activity to produce derivatives of C3b that now specifically engage additional receptors. Likewise, C1-Inh leads to C1q remaining on the immune complex to interact with the C1q receptor. Thus the complement system is designed to allow rapid, efficient, unimpeded activation on an appropriate foreign target while regulatory proteins intervene to prevent three undesirable consequences of complement activation: excessive activation on a single target, fluid phase activation, and activation on self.

Anaphylatoxins↗
PubMed1996

The HeLa cell receptor for enterovirus 70 is decay-accelerating factor (CD55).

Enterovirus 70 (EV70) is a recently emerged human pathogen belonging to the family Picornaviridae. The ability of EV70 to infect a wide variety of nonprimate cell lines in vitro is unique among human enteroviruses. The importance of virus receptors as determinants of viral host range and tropism led us to study the host cell receptor for this unusual picornavirus. We produced a monoclonal antibody (MAb), EVR1, which bound to the surface of HeLa cells and protected them against infection by EV70 but not by poliovirus or by coxsackievirus B3. This antibody also inhibited the binding of [35S]EV70 to HeLa cells. MAb EVR1 did not bind to monkey kidney (LLC-MK2) cells, nor did it protect these cells against virus infection. In Western immunoassays and in immunoprecipitations, MAb EVR1 identified a HeLa cell glycoprotein of approximately 75 kDa that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Decay-accelerating factor (DAF, CD55) is a 70- to 75-kDa GPI-anchored membrane protein that is involved in the regulation of complement and has also been shown to function as a receptor for several enteroviruses. MAb EVR1 bound to Chinese hamster ovary (CHO) cells constitutively expressing human DAF. Anti-DAF MAbs inhibited EV70 binding to HeLa cells and protected them against EV70 infection. Transient expression of human DAF in murine NIH 3T3 cells resulted in binding of labelled EV70 and stably, transformed NIH 3T3 cells expressing DAF were able to support virus replication. These data indicate that the HeLa cell receptor for EV70 is DAF.

3T3 Cells↗
PubMed1995

Identification of complement regulatory domains in human factor H.

Factor H, a regulator of complement activation, contains 20 short consensus repeat (SCR) domains common among the family of C3b/C4b-binding proteins. Chinese hamster ovary cells transfected with cDNA corresponding to the N-terminal tryptic fragment of factor H (containing SCR 1-5 and part of SCR 6) secreted protein with cofactor activity for factor I-dependent cleavage of C3b. A series of deletion mutants, each lacking one of the first five SCR, were constructed, and the supernatants of transfected Chinese hamster ovary cells were tested for cofactor activity. Supernatants of Chinese hamster ovary cells transfected with SCR 1, SCR 4, and SCR 5 deletion mutants retained cofactor function, although the SCR 1 deletion had reduced cofactor activity. Deletion of SCR 2 or 3 totally abolished cofactor activity. Expression and functional analysis of SCR units 1-3, 2-3, and 2-4 demonstrated that the SCR 1-3 unit is sufficient for cofactor activity, but SCR 1-4 is required for full activity. For assays involving cell protection, a construct linking SCR 1-5 to the glycosyl-phosphatidylinositol anchor of decay-accelerating factor was prepared, and stable transfectants were obtained. These cells were protected against complement-mediated cytotoxicity, similarly to decay-accelerating factor- and membrane cofactor protein-transfected cells. These studies define the complement regulatory domains in factor H and suggest that the general complement functional unit for C3 convertase regulation involves three or four consecutive SCR units.

Animals↗
PubMed1995

Distinct tyrosine kinase activation and Triton X-100 insolubility upon Fc gamma RII or Fc gamma RIIIB ligation in human polymorphonuclear leukocytes. Implications for immune complex activation of the respiratory burst.

Two tyrosine kinase-dependent pathways exist for activation of the respiratory burst by polymorphonuclear leukocyte (PMN) immunoglobulin G Fc receptors. Direct ligation of Fc gamma RII activates the respiratory burst, but ligation of the glycan phosphoinositol-linked Fc gamma RIIIB does not. Instead, this receptor and the integrin complement receptor CR3 synergize in activation of the respiratory burst (Zhou, M.-J., and Brown, E. J. (1994) J. Cell Biol. 125, 1407-1416). Here we show that direct ligation of Fc gamma RII leads to activation and Triton X-100 insolubility of the Src family kinase Fgr, without effect on the related myeloid Src family member Hck. In contrast, adhesion of PMN via Fc gamma RIIIB leads to activation and Triton X-100 insolubility of Hck but not Fgr. The exclusive association of Fc gamma RIIIB with Hck activation and Triton insolubility is not solely a result of its glycan phosphoinositol anchor, since decay accelerating factor (CD55), another prominent glycan phosphoinositol-anchored PMN protein, is associated with Fgr insolubility to a greater extent than Hck. Ligation of decay accelerating factor, with or without coligation of CR3, does not activate the PMN respiratory burst. Coligation of Fc gamma RIIIB with Fc gamma RII overcomes the pertussis toxin inhibition of H2O2 production in response to direct ligation of Fc gamma RII. These data support the hypothesis that activation of Hck upon Fc gamma RIIIB ligation has a role in generation of the synergistic respiratory burst.

Antigen-Antibody Complex↗
PubMed1995

Caveolin is palmitoylated on multiple cysteine residues. Palmitoylation is not necessary for localization of caveolin to caveolae.

Caveolae are subdomains of the plasma membrane which concentrate cholesterol, glycosphingolipids, and glycosylphosphatidylinositol-linked proteins. It has recently been demonstrated that specific members of the Src family of protein tyrosine kinases require palmitoylation of NH2-terminal cysteine residues to localize in caveolae. Here we report that caveolin, an integral membrane protein which forms part of the coat of caveolae, also incorporates palmitate through linkage to cysteine residues. Caveolin contains only three cysteine residues which are all located on the COOH-terminal side of the hydrophobic transmembrane region. Immunofluorescent staining of cells transfected with caveolin indicated that, like the NH2 terminus, this COOH-terminal region is located on the cytoplasmic side of the plasma membrane. Studies of cysteine substitution mutants showed that all three cysteines are capable of incorporating palmitate and that the juxtamembrane Cys133 residue is the predominant site of palmitoylation. Simultaneous mutation of all three cysteine residues in caveolin resulted in the loss of ability to incorporate palmitate; however, this did not affect localization of the protein. Thus, palmitoylation of cysteine residues in nonmembrane spanning Src family protein tyrosine kinases has different consequences than in the transmembrane protein caveolin.

Amino Acid Sequence↗
PubMed1995

Measles virus and C3 binding sites are distinct on membrane cofactor protein (CD46).

The human complement regulatory protein membrane cofactor protein (CD46) is the cellular receptor for measles virus (MV), whereas decay accelerating factor (DAF; CD55), a structurally similar complement regulatory protein, does not bind MV. To characterize the interaction between MV and CD46, mutants of the CD46 protein and hybrid molecules between CD46 and DAF were tested for their ability to act as MV receptors. The transmembrane domain and cytoplasmic tail of CD46 were not required for receptor function as cells expressing the CD46 extracellular domain linked to the glycosyl-phosphatidylinositol tail of DAF were rendered susceptible to MV infection. Chimeric proteins exchanging the four extracellular short consensus repeat (SCR) domains between CD46 and DAF indicated that only molecules with both SCR1 and SCR2 from CD46 allowed a productive MV infection. Further, monoclonal antibodies (mAbs) against SCR1 or SCR2 of CD46 blocked MV infection, whereas a mAb against SCR3 and SCR4 did not. The latter mAb blocks C3b/C4b binding (which maps to SCR3 and SCR4) whereas the former mAbs do not. Thus, our data indicate that both SCR1 and SCR2 make up the MV receptor determinant in CD46. These results also suggest avenues for development of therapeutic agents to inhibit MV binding and thus infection and disease.

Animals↗
PubMed1995

Amino-terminal palmitate or polybasic domain can provide required second signal to myristate for membrane binding of p56lck.

Recent work has shown that several members of the src family of protein tyrosine kinases (PTKs) are modified by palmitoylation, including p56lck and p59fyn but not p60src. Mapping of the sites of palmitoylation in p56lck identified cys3 as the major site and cys5 as a minor site of palmitoylation. A non-palmitoylated p56lck(cys3,5-->ser) mutant was localized exclusively in the cytoplasm despite the presence of amino-terminal myristoylation, thus indicating that palmitoylation of p56lck was necessary for membrane binding. The addition of a domain of six lysine residues to a non-palmitoylated p56lck mutant was sufficient to re-establish membrane binding but not to target the non-palmitoylated p56lck to caveolae. These results establish that two signals, myristoylation plus either palmitoylation or a polybasic domain, are necessary for membrane binding of src family PTKs.

Amino Acid Sequence↗
PubMed1995

dra-related X adhesins of gestational pyelonephritis-associated Escherichia coli recognize SCR-3 and SCR-4 domains of recombinant decay-accelerating factor.

Bacterial adhesins are important virulence factors that allow colonization of the human urogenital tract by Escherichia coli. Adhesins of the Dr family have been found to be more frequently expressed in strains associated with symptomatic urinary tract infections. Because of the high frequency of symptomatic urinary tract infections during pregnancy, we screened E. coli isolates from 64 gestational pyelonephritis patients for the expression of Dr and X adhesins to address their potential virulence roles in this population. Using PCR and primers for the afaB gene, we detected dra-related operons in 17 isolates (27%). On the basis of the lack of hemagglutination of Dr(a-) erythrocytes containing a point mutation in the decay-accelerating factor (DAF) short consensus repeat-3 (SCR-3) domain, 12 of these strains were categorized as classical Dr adhesins. The hemagglutination of O erythrocytes by Dr+ strains was blocked or reduced by a monoclonal antibody to the DAF SCR-3 domain. The remaining five dra-positive strains agglutinated Dr(a-) erythrocytes. Monoclonal antibody to the DAF SCR-3 domain failed to block O-erythrocyte hemagglutination. Adhesins in these strains did not fulfill criteria for Dr hemagglutinins because of the undefined receptor specificities and were categorized as X. E. coli strains bearing dra-related X adhesins bound to DAF cDNA-transfected Chinese hamster ovary cells. Three of these dra-related X-adhesin-bearing E. coli strains failed to attach to the SCR-3 delta deletion transfectant, which suggested that binding sites were located in the SCR-3 domain but outside the region blocked by the monoclonal anti-SCR-3 immunoglobulin G. The binding sites of the remaining two dra-related X adhesin strains were localized to the SCR-4 domain, as the attachment was shown to be abolished on an SCR-4 delta mutant but unaffected by an SCR-3 delta deletion. The heterogeneity in the binding sites of E. coli DAF (Dr) family adhesins from gestational pyelonephritis isolates may reflect the ability of the adhesins to evolve to recognize alternate peptide epitopes for efficient colonization.

Adhesins, Escherichia coli↗
PubMed1995

Coxsackievirus B3 adapted to growth in RD cells binds to decay-accelerating factor (CD55).

A coxsackievirus B3 (CB3) isolate adapted to growth in RD cells shows an alteration in cell tropism as a result of its capacity to bind a 70-kDa cell surface molecule expressed on these cells. We now show that this molecule is the complement regulatory protein, decay-accelerating factor (DAF) (CD55). Anti-DAF antibodies prevented CB3 attachment to the cell surface. Radiolabeled CB3 adapted to growth in RD cells bound to CHO cells transfected with human DAF, whereas CB3 (strain Nancy), the parental strain, did not bind to DAF transfectants. These results indicate that growth of CB3 in RD cells selected for a virus strain that uses DAF for cell surface attachment.

Antibodies, Monoclonal↗