Molecular characterization of two monoclonal antibodies specific for the LDL receptor-binding site of human apolipoprotein E

Apolipoprotein E (apoE), a 299 amino acid protein, is a ligand for the low density lipoprotein receptor (LDLr). It has been established that basic amino acids situated between apoE residues 136 and 150 participate in the interaction of apoE with the LDLr. Evidence suggests that apoE is heterogeneous on lipoproteins in its conformation and in its ability to react with cell surface receptors. Our goal was to produce mAbs that could serve as conformational probes of the LDLr binding site of apoE. We used a series of apoE variants that have amino acid substitutions at residues 136, 140, 143, 144, 145, 150, 152, and 158 to identify the epitopes of two anti-human apoE monoclonal antibodies (mAbs), 1D7 and 2E8, that inhibit apoE-mediated binding to the LDLr. We show that most of the variants that have reduced reactivity with the LDL receptor also have reduced reactivity with the mAbs. The epitopes for both mAbs appear to include residues 143 through 150 and thus coincide with the LDLr-binding site of apoE. It is notable that mAb 2E8, but not 1D7, resembles the LDLr in showing a reduced reactivity with apoE (Arg158 --> Cys). While most of the receptor-defective variants involve replacement of apoE residues directly implicated in binding, substitution of Arg158 by Cys is thought to indirectly affect binding of apoE to the LDLr by altering the conformation of the receptor-binding site. To determine whether the similarity in specificities of the mAbs and the LDLr reflect structural similarities, we cloned and characterized the cDNAs encoding the light and heavy chains of both mAbs. Primary sequence analysis revealed that, although these two antibodies react with overlapping epitopes, their respective complementarity determining regions (CDRs) share little homology, especially those of their heavy chains. The two mAbs, therefore, likely recognize different epitopes or topologies within a limited surface of the apoE molecule. Four negatively charged amino acids were present in the second CDR of the 2E8 heavy chain that could be approximately aligned with acidic amino acids within the consensus sequence of the LDLr ligand-binding domain. This could indicate that mAb 2E8 and the LDLr use a common mode of interaction with apoE.     

A novel LFA-1 activation epitope maps to the I domain

A panel of 21 alpha-subunit (CD11a) and 10 beta-subunit (CD18) anti-LFA-1 mAbs was screened for ability to activate LFA-1. A single anti-CD11a mAb, MEM-83, was identified which was able to directly induce the binding of T cells to purified ICAM-1 immobilized on plastic. This ICAM-1 binding could be achieved by monovalent Fab fragments of mAb MEM-83 at concentrations equivalent to whole antibody, was associated with appearance of the "activation reporter" epitope detected by mAb 24, and was completely inhibited by anti-ICAM-1 and LFA-1 blocking mAbs. The epitope recognized by mAb MEM-83 was distinct from that recognized by mAb NKI-L16, an anti-CD11a mAb previously reported to induce LFA-1 activation, in that it was constitutively present on freshly isolated peripheral blood mononuclear cells and was not divalent cation dependent for expression. The ICAM-1 binding activity induced by mAb MEM-83 was, however, dependent on the presence of Mg2+ divalent cations. Using an in vitro-translated CD11a cDNA deletion series, we have mapped the MEM-83 activation epitope to the "I" domain of the LFA-1 alpha subunit. These studies have therefore identified a novel LFA-1 activation epitope mapping to the I domain of LFA-1, thereby implicating this domain in the regulation of LFA-1 binding to ICAM-1.     

Delineation of tyrosine-containing epitopes within the beta subunit of bovine thyrotropin

The epitopes recognised by two monoclonal antibodies (mAb 279 and mAb 299), specific for the beta subunit of bovine thyroid-stimulating hormone (bTSH), have been localised using a technique in which the tyrosine residues in the bTSH beta subunit were subjected to modification when the bTSH beta subunit was complexed with either mAb or in the free, unbound state. The epitope recognised by mAb 279 was localised to the C-terminal region of bTSH beta with the tyrosine residue Tyr104 protected from modification by the presence of this mAb. In addition, the experimental results indicate that the tyrosine residues Tyr18 and/or Tyr112 are also involved in the mAb 279 epitope. The epitope recognised by mAb 299 was localised to the region 59-74 of bTSH beta as both Tyr59 and Tyr74 were protected from modification by the presence of this mAb. Since both mAbs have been previously found to inhibit receptor binding, the sequence regions/amino acid positions recognised by these mAbs are likely to represent determinants for receptor binding. Moreover, these data indicate that the identified amino acid residues are located on the surface of the molecule, consistent with predictions of the tertiary structure of the bTSH beta subunit based on the recently elucidated X-ray crystal structure of human chorionic gonadotropin.     

The amino-terminal region of Tyk2 sustains the level of interferon alpha receptor 1, a component of the interferon alpha/beta receptor

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-alpha (IFN-alpha) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-alpha. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-alpha receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficient in vitro IFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-alpha. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.     

Interaction of chemokine receptor CCR5 with its ligands: multiple domains for HIV-1 gp120 binding and a single domain for chemokine binding

CCR5 is a chemokine receptor expressed by T cells and macrophages, which also functions as the principal coreceptor for macrophage (M)-tropic strains of HIV-1. To understand the molecular basis of the binding of chemokines and HIV-1 to CCR5, we developed a number of mAbs that inhibit the various interactions of CCR5, and mapped the binding sites of these mAbs using a panel of CCR5/CCR2b chimeras. One mAb termed 2D7 completely blocked the binding and chemotaxis of the three natural chemokine ligands of CCR5, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta, to CCR5 transfectants. This mAb was a genuine antagonist of CCR5, since it failed to stimulate an increase in intracellular calcium concentration in the CCR5 transfectants, but blocked calcium responses elicited by RANTES, MIP-1alpha, or MIP-1beta. This mAb inhibited most of the RANTES and MIP-1alpha chemotactic responses of activated T cells, but not of monocytes, suggesting differential usage of chemokine receptors by these two cell types. The 2D7 binding site mapped to the second extracellular loop of CCR5, whereas a group of mAbs that failed to block chemokine binding all mapped to the NH2-terminal region of CCR5. Efficient inhibition of an M-tropic HIV-1-derived envelope glycoprotein gp120 binding to CCR5 could be achieved with mAbs recognizing either the second extracellular loop or the NH2-terminal region, although the former showed superior inhibition. Additionally, 2D7 efficiently blocked the infectivity of several M-tropic and dual-tropic HIV-1 strains in vitro. These results suggest a complicated pattern of HIV-1 gp120 binding to different regions of CCR5, but a relatively simple pattern for chemokine binding. We conclude that the second extracellular loop of CCR5 is an ideal target site for the development of inhibitors of either chemokine or HIV-1 binding to CCR5.     

Characterization of the CCR2 chemokine receptor: functional CCR2 receptor expression in B cells

We have derived anti-human CCR2-specific mAbs by immunization with synthetic peptides corresponding to CCR2 sequences presumably involved in the interaction with its ligand(s). The characterization of these mAbs includes the ability to recognize the CCR2 receptor specifically, as well as the function based on their ability to promote Ca2+ influx or to block MCP-1-induced Ca2+ influx and chemotaxis. One mAb (MCP-1 R02) that is directed to the NH2 terminal domain of the CCR2 receptor has MCP-1 agonist activity, and two that recognize the third extracellular domain (MCP-1R04 and MCP-1 R05) have MCP-1 antagonist activity. We analyzed the presence of CCR2 in several PBL and tonsil-derived leukocyte populations and found expression of this receptor in monocytes, activated T cells, and, surprisingly, in B cells. CCR2 receptor expression in B cells was further corroborated in Southern blot using CCR2-specific probes. Moreover, both MCP-1 and the agonist mAb trigger specific B cell migration via a PTX-sensitive mechanism, indicating the presence of a functional CCR2 receptor in these cells.     

N-terminal and central regions of the human CD44 extracellular domain participate in cell surface hyaluronan binding

CD44 molecules are cell surface receptors for hyaluronan (HA). To define regions of the extracellular domain of CD44 that are important for HA binding, we have studied the ability of HA-blocking CD44 mAbs to bind to CD44 from a variety of sources. Five CD44 mAbs (5F12, BRIC235, 3F12, BU-75, and HP2/9) of 21 studied were identified that at least partially blocked FITC-labeled HA (HA-FITC) binding to the standard form of CD44 (CD44S) in CD44-transfected Jurkat cells. Analysis of reactivity of HA-blocking CD44 mAbs defined three distinct epitopes. Lack of reactivity of mAb 5F12 with a CD44 fusion protein (CD44-Rg) containing an N-terminal truncation of 20 amino acids (aa), as well as reactivity of mAb 5F12 with an N-terminal CD44 synthetic peptide (CD44-9A), demonstrated that the N-terminal proximal region of CD44 (aa 1 to 20) was involved in mAb 5F12 binding. A mutant cell line, CEM-NKR, derived from the T-ALL cell line, CEM, did not bind mAb 5F12 nor bind HA, whereas wild-type CEM did bind mAb 5F12 and HA. Sequence analysis of wild-type CEM and CEM-NKR CD44 cDNA demonstrated a G to A point mutation at position 575 in the CD44 cDNA of CEM-NKR, resulting in an arginine to histidine mutation at aa position 154. Taken together, our studies demonstrated that there are three epitopes to which HA-blocking mAbs bind in the extracellular domain of CD44, and that the CD44 N-terminal proximal and central regions are two regions in the extracellular domain of CD44 that may interact and either mediate or regulate HA binding to cell surface CD44.     

Accessibility of epitopes on UvrB protein in intermediates generated during incision of UV-irradiated DNA by the Escherichia coli Uvr(A)BC endonuclease

Structural intermediates generated during incision of damaged DNA by the Uvr(A)BC endonuclease were probed with monoclonal antibodies (mAbs) raised against the Escherichia coli UvrB protein. It was found that the epitope of B2C5 mAb, mapped at amino acids (aa) 171-278 of UvrB, is not accessible in any of the preformed Uvr intermediates. Preformed B2C5-UvrB immunocomplexes, however, inhibited formation of those intermediates. B2C5 mAb seems to interfere with the formation of the UvrA-UvrB complex due to overlapping of its epitope and the UvrA binding region of UvrB. Conversely, the epitope of B3C1 mAb (aa 1-7 and/or 62-170) was accessible in all Uvr intermediates. The epitope of B*2E3 mAb (aa 171-278) was not accessible in any of the nucleoprotein intermediates preceding UvrB-DNA preincision complex. However, B*2E3 was able to immunoprecipitate this complex and to inhibit overall incision. B2A1 mAb (aa 8-61) inhibited formation of those Uvr intermediates requiring ATP binding and/or hydrolysis by UvrB. B*2B9 mAb (aa 473-630) inhibited Uvr nucleoprotein complexes involving UvrB. B*2B9 seems to prevent the binding of the UvrA-UvrB complex to DNA. The epitope of the B*3E11 mAb (aa 379-472) was not accessible in Uvr complexes formed at damaged sites. These results are discussed in terms of structure-functional mapping of UvrB protein.     

A negative regulatory region in the intracellular domain of the human interferon-alpha receptor

Interferon-alpha (IFN-alpha)-mediated intracellular signaling is initiated by ligand-induced receptor dimerization, tyrosine phosphorylation of the Tyk2 and Jak1 tyrosine kinases, and subsequent phosphorylation of the Stat1 and Stat2 proteins. The IFN-alpha receptor consists of at least two distinct subunits. One subunit, IFNAR1, has low affinity binding for interferon yet is required for signal transduction. We introduced mutations in the cytoplasmic domain of human IFNAR1 in order to identify residues involved in the mediation of biological responses. We took advantage of the species specificity of the interferon receptors by analyzing human IFN-alpha-induced major histocompatibility complex class I antigen expression in mouse L929 cells stably transfected with mutant human receptors. The membrane proximal 60-amino acids were insufficient to signal a biological response even though within these residues Tyk2 and Stat2 binding sites have been identified. IFN-alpha-induced receptor tyrosine phosphorylation was not critical for signaling because mutation of Tyr residues to Phe did not prevent the biological response to IFN-alpha. The deletion of a 16-amino acid region highly homologous between species created a receptor which signals an enhanced response. Tyrosine dephosphorylation is a component of this enhanced response as mutation of the Tyr residues within this region to Phe resulted in a receptor with increased sensitivity to IFN. The known signaling molecules that interact with IFNAR1 are positive regulators of IFN-alpha function. The presence of this domain in the COOH-terminal region suggests that the receptor may interact with signaling molecules that negatively regulate interferon responses.     

Identification of amino acid residues that form part of the ligand-binding pocket of integrin alpha5 beta1

Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA) is a novel ligand peptide for integrin alpha5 beta1, which blocks alpha5 beta1-mediated cell adhesion to fibronectin (Koivunen, E., Wang, B., and Ruoslahti, E. (1994) J. Cell Biol. 124, 373-380). Here we have localized the binding site for RRETAWA on alpha5 beta1 using inhibitory monoclonal antibodies (mAbs) and site-directed mutagenesis. A cyclic peptide containing this sequence (*CRRETAWAC*) had little effect on the binding of most anti-alpha5 and anti-beta1 mAbs to alpha5 beta1 but completely blocked binding of the anti-alpha5 mAb 16 in a directly competitive manner. Hence, the binding site of RRETAWA appears to closely overlap with the epitope of mAb 16. *CRRETAWAC* also acted as a direct competitive inhibitor of the binding of Arg-Gly-Asp (RGD)-containing fibronectin fragments to alpha5 beta1, suggesting that the binding site for RRETAWA is also closely overlapping with that for RGD. However, differences between the binding sites of RRETAWA and RGD were apparent in that (i) RGD peptides allosterically inhibited the binding of mAb 16 to alpha5 beta1, and (ii) several mAbs that perturbed binding of alpha5 beta1 to RGD had little effect on binding of alpha5 beta1 to RRETAWA. A double mutation in alpha5 (S156G/W157S) blocked the interaction of both RRETAWA and mAb 16 with alpha5 beta1 but had no effect on fibronectin binding or on the binding of other anti-alpha5 mAbs. Ser156-Trp157 is located near the apex of a putative loop region on the upper surface of a predicted beta-propeller structure formed by the NH2-terminal repeats of alpha5. Our findings suggest that this sequence forms part of the ligand-binding pocket of alpha5 beta1. Furthermore, as Ser156-Trp157 is unique to the alpha5 subunit, it may be responsible for the specific recognition of RRETAWA by alpha5 beta1.     

HLA (A*0201) mimicry by anti-idiotypic monoclonal antibodies

Soluble MHC Ags and anti-Id (anti-anti-MHC) Abs have both been shown to inhibit MHC alloantigen-specific B cell responses in vivo. We hypothesized that some anti-idiotypic Abs function as divalent molecular mimics of soluble HLA alloantigen. To test this idea, we studied two well-defined anti-idiotypic mAbs, T10-505 and T10-938, elicited in syngeneic BALB/c mice by immunization with CRll-351, an HLA-A2,24,28-specific mAb. Each anti-Id induced "Ab-3" Abs in rabbits that cross-reacted with HLA-A2 but not with HLA-B Ags. Furthermore, each anti-Id could bind to and block Ag recognition by Ha5C2.A2, a human homologue of mAb CRll-351. Both anti-Id mAb displayed weak reactivity with the human mAb SN66E3, which recognized an overlapping but distinct determinant of HLA-A2 Ags; neither reacted with human mAb MBW1, which recognized a nonoverlapping HLA-A2 determinant. Amino acid sequence comparison of mAb CRII-351 heavy and light chain variable region complementarity-determining regions (CDRs) with those of mAb Ha5C2.A2 and SN66E3 revealed short regions of homology with both human mAb; a large insert in the light chain CDR1 of mAb SN66E3 distinguished it from both CRll-351 and Ha5C2.A2. The amino acid sequences of mAb T10-505 and T10-938, which differed markedly from each other, revealed no homology to the alpha2 domain sequence of HLA-A*0201 that contains the CRll-351 mAb-defined epitope. We conclude that structurally different anti-Id Abs can mimic a polymorphic conformational epitope of an HLA Ag. In the case of T10-505 and T10-938 mimicry was not based on exact replication of the epitope by the hypervariable loops of the anti-Id mAb.     

Differences in activity between alpha and beta type I interferons explored by mutational analysis

Type I interferon (IFN) subtypes alpha and beta share a common multicomponent, cell surface receptor and elicit a similar range of biological responses, including antiviral, antiproliferative, and immunomodulatory activities. However, alpha and beta IFNs exhibit key differences in several biological properties. For example, IFN-beta, but not IFN-alpha, induces the association of tyrosine-phosphorylated receptor components ifnar1 and ifnar2, and has activity in cells lacking the IFN receptor-associated, Janus kinase tyk2. To define the structural basis for these functional differences we produced human IFN-beta with point mutations and compared them to wild-type IFN-beta in assays that distinguish alpha and beta IFN subtypes. IFN-beta mutants with charged residues (N86K, N86E, or Y92D) introduced at two positions in the C helix lost the ability to induce the association of tyrosine-phosphorylated receptor chains and had reduced activity on tyk2-deficient cells. The combination of negatively charged residues N86E and Y92D (homologous with IFN-alpha8) increased the cross-species activity of the mutant IFN-betas on bovine cells to a level comparable to that of human IFN-alphas. In contrast, point mutations in the AB loop and D helix had no significant effect on these subtype-specific activities. A subset of these latter mutations did, however, reduce activity in a manner analogous to IFN-alpha mutations. The effects of these mutations on IFN-beta activity are discussed in the context of a family of related ligands acting through a common receptor and signaling pathway.     

Surgical treatment of obstructive jaundice

Genetically engineered structural variants of human beta2-microglobulin (beta2m) were produced by sequence exchange with mouse beta2m for the purpose of examining species-specific antigenic determinant expression. For aggregate mapping, mouse and human beta2m, which differ by 30% in their primary sequence of 99 amino acids, were prepared as chimeric (human X mouse) molecules and expressed in the FO-1 beta2m-null human melanoma cell line. A chimera containing residues 1-69 from human beta2m (and residues 70-99 from mouse beta2m) induced expression of the epitopes defined by the anti-beta2m monoclonal antibodies (mAb) BBM.1, NAMB-1, and L368; the reverse chimera did not, although HLA class I heavy chain was evident on the cell surface as determined with the TP25.99 mAb. For fine dissection of the epitopes defined by these mAbs, site-directed mutants of beta2m were prepared by replacement of individual amino acids in human beta2m with the dimorphic residue from mouse beta2m. Substitutions were made at each divergent residue between positions 1 and 66 and, as controls for COOH-terminal modification, a series of residues between positions 75 and 94. Replacement of amino acids 38, 44, and 45, but not 16 other dimorphic residues in the linear stretch from residue 1 to residue 66, resulted in the loss of, or gross reduction in, binding by mAbs BBM.1 and NAMB-1. A reduction in binding was also observed for mAb L368. These data provide strong evidence that the antigenic epitopes defined by these mAb map to a region including S3 and its adjacent intra-beta-strand turn of the three-stranded beta-pleated sheet of beta2m. The mapping of these epitopes is consistent with their accessibility in the assembled major histocompatibility complex class I molecule and indicates that the region from amino acid 38 to 45 is an important structural feature in the "foreignness" of human and mouse beta2m.     

The I domain of integrin LFA-1 interacts with ICAM-1 domain 1 at residue Glu-34 but not Gln-73

Using a solid phase assay, we show that isolated LFA-1 I domain binds ICAM-1 in a Mg2+-dependent manner and is blocked by anti-I domain monoclonal antibodies. This activity mirrors that of the intact receptor (Dransfield, I., Caba?as, C., Craig, A., and Hogg, N. (1992) J. Cell Biol. 116, 219-226) and suggests that the I domain controls divalent cation-dependent receptor function. In ICAM-1, domain 1 residues Glu-34 and Gln-73 have been identified as critical for binding of LFA-1 as an intact receptor (Staunton, D. E., Dustin, M. L., Erickson, H. P., and Springer, T. A. (1990) Cell 61, 243-254). For the first time, we show that isolated I domain binds to domain 1 of ICAM-1 and that this interaction is inhibited partially by mutation of Glu-34 but not by Gln-73. The anti-ICAM-1 monoclonal antibody RR1/1, which maps to Gln-73 (Staunton, D. E., Dustin, M. L., Erickson, H. P., and Springer, T. A. (1990) Cell 61, 243-254), enhances I domain binding, suggesting potential allosteric control or coordinate binding by this region. Finally, I domain binding inhibited by Glu-34 ICAM-1 mutation correlates with divalent cation dependence, indicating that this residue might be in direct contact with the metal ion-dependent adhesion site. Thus, we describe the interaction between the LFA-1 I domain and ICAM-1, an event that controls the function of the intact receptor but includes only part of the complete ligand binding site.     

Role of malignant hyperthermia domain in the regulation of Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum

A fusion protein encompassing Gly341 of the skeletal muscle ryanodine receptor was used to raise monoclonal antibodies; epitope mapping demonstrates that monoclonal antibody 419 (mAb419) reacts with a sequence a few residues upstream from Gly341. The mAb419 was then used to probe ryanodine receptor (RYR) functions. Our results show that upon incubation of triads vesicles with mAb419 the Ca2+-induced Ca2+ release rate at pCa 8 was increased. Equilibrium evaluation of [3H]ryanodine binding at different [Ca2+] indicates that mAb419 shifted the half-maximal [Ca2+] for stimulation of ryanodine binding to lower value (0.1 versus 1.2 microM). Such functional effects may be due to a direct action of the Ab on the Ca2+ binding domain of the RYR or to the perturbation by the Ab of the intramolecular interaction between the immunopositive region and regulatory domain of the RYR. The latter hypothesis was tested directly using the optical biosensor BIAcore (Pharmacia Biotech Inc.): we show that the immunopositive RYR polypeptide is able to interact with the native RYR complex. Ligand overlays with immunopositive digoxigenin-RYR fusion protein indicate that such an interaction might occur with a calmodulin binding domain (defined by residues 3010-3225) and with a polypeptide defined by residues 799-1172. In conclusion our results suggest that the stimulation by the mAb419 of the RYR channel activity is due to the perturbation of an intramolecular interaction between the immunopositive polypeptide and a Ca2+ regulatory site probably corresponding to a calmodulin binding domain.     

The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D

The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR/PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1. 302 to HIgR/PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR/PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR/PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for in vitro physical interaction with gD. The in vitro binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.     

Expression cloning of a rat liver Na(+)-independent organic anion transporter

The interactions between platelets and plasma proteins previously shown to adhere to biomaterials were evaluated, using monoclonal antibodies (mAbs) against specific platelet surface glycoprotein (GP) receptors. Purified 51Cr-labeled human platelets in plasma-free medium were incubated with each of the following antibodies: mAb 10E5 [anti-GP IIb/IIIa; fibrinogen, von Willebrand factor (vWF), and fibronectin receptor]; mAb 6D1 (anti-GP Ib-IX; vWF receptor); mAb IV.3 (anti-Fc gamma RII; IgG receptor); polyclonal antiserum A108 or mAb BIIG4 (anti-GP Ic-IIa; fibronectin receptor). Antibody-treated platelets were added to microtiter wells coated with fibronectin, fibrinogen, vWF, IgG, vitronectin, albumin, or platelet-poor plasma (PPP). 51Cr-labeled platelet adhesion to matrix proteins was expressed as a percentage of that measured on PPP-coated surface. Platelets adhered to fibrinogen, fibronectin, vWF, or IgG immobilized on polystyrene. Limited binding to either vitronectin or albumin was detected. Binding to fibrinogen and IgG was blocked by mAb 10E5. Binding to IgG was also blocked by mAb IV.3. Binding to fibronectin, reduced in the presence of mAb 10E5, mAb BIIG4, or the polyclonal antiserum A108 alone, was further reduced by combined 10E5 and BIIG4 or 10E5 and A108. Neither mAb 10E5 nor 6D1 alone blocked adhesion to vWF; however, the combination of 10E5 and 6D1 significantly reduced platelet adhesion to this matrix. Finally, platelet adhesion to the plasma-coated surface was reduced by mAbs 10E5 and BIIG4. These results indicate that multiple adhesion receptors can mediate platelet adhesion to matrix proteins immobilized on surfaces.     

Monoclonal antibodies to the extracellular domain of prostate-specific membrane antigen also react with tumor vascular endothelium

Prostate-specific membrane antigen (PSMA), initially defined by monoclonal antibody (mAb) 7E11, is a now well-characterized type 2 integral membrane glycoprotein expressed in a highly restricted manner by prostate epithelial cells. 7E11 has been shown to bind an intracellular epitope of PSMA that, in viable cells, is not available for binding. Herein, we report the initial characterization of the first four reported IgG mAbs that bind the external domain of PSMA. Competitive binding studies indicate these antibodies define two distinct, noncompeting epitopes on the extracellular domain of PSMA. In contrast to 7E11, these mAbs bind to viable LNCaP cells in vitro. In addition, they show strong immunohistochemical reactivity to tissue sections of prostate epithelia, including prostate cancer. These mAbs were also strongly reactive with vascular endothelium within a wide variety of carcinomas (including lung, colon, breast, and others) but not with normal vascular endothelium. These antibodies should prove useful for in vivo targeting to prostate cancer, as well as to the vascular compartment of a wide variety of carcinomas.     

Priming of blood neutrophils in children with cystic fibrosis: correlation between functional and phenotypic expression of opsonin receptors before and after platelet-activating factor priming

Ovine interferon-tau (ovIFN-tau) is a pregnancy recognition hormone required for normal embryonic development in sheep. In addition to its novel role in reproductive physiology, ovIFN-tau displays antiviral and antiproliferative activities similar to the IFN-alpha subtypes. To probe the structural basis for its unique activity profile, the crystal structure of ovIFN-tau has been determined at 2.1 A resolution. The fold of ovIFN-tau is similar to the previously determined crystal structures of human IFN-alpha2b and human and murine IFN-beta, which each contain five alpha-helices. Comparison of ovIFN-tau with huIFN-alpha2b, huIFN-beta, and muIFN-beta reveals unexpected structural differences that occur in regions of considerable sequence identity. Specifically, main-chain differences up to 11 A occur for residues in helix A, the AB loop, helix B, and the BC loop. Furthermore, these regions are known to be important for receptor binding and biological activity. Of particular interest, a buried ion pair is observed in ovIFN-tau between Glu71 and Arg145 which displaces a conserved tryptophan residue (Trp77) from the helical bundle core. This ion pair represents a major change in the core of ovIFN-tau compared to huIFN-alpha2b. Based on amino acid sequence comparisons, these ovIFN-tau structural features may be conserved in several human IFN-alpha subtypes and IFN-omega. The structure identifies potential problems in interpreting site-directed mutagenesis data on the human IFN-alpha family that consists of 12 proteins.