Direct measurement of antigen binding properties of CD1 proteins using fluorescent lipid probes

CD1 proteins are antigen-presenting molecules that bind foreign and self-lipids and stimulate specific T cell responses. In the current study, we investigated ligand binding by CD1 proteins by developing a fluorescent probe binding approach using soluble recombinant human CD1 proteins. To increase stability and yield, soluble group 1 CD1 (CD1b and CD1c) and group 2 CD1 (CD1d) proteins were produced as single chain secreted CD1 proteins in which beta2-microglobulin was fused to the N termini of the CD1 heavy chains by a flexible peptide linker sequence. Analysis of ligand binding properties of single chain secreted CD1 proteins by using fluorescent lipid probes indicated significant differences in ligand preference and in pH dependence of binding by group 1 versus group 2 CD1 proteins. Whereas group 1 CD1 isoforms (CD1b and CD1c) show stronger binding of nitrobenzoxadiazole (NBD)-labeled dialkyl-based ligands (phosphatidylcholine, sphingomyelin, and ceramide), group 2 CD1 (CD1d) proteins were stronger binders of small hydrophobic probes such as 1-anilinonaphthalene-8-sulfonic acid and 4,4'-dianilino-1,1'-naphthyl-5,5'-disulfonic acid. Competition studies indicated that binding of fluorescent lipid probes involved association of the probe with the hydrophobic ligand binding groove of CD1 proteins. Analysis of selected alanine substitution mutants of human CD1b known to inhibit antigen presentation showed that NBD-labeled lipid probe binding could be used to distinguish mutations that interfere with ligand binding from those that affect T cell receptor docking. Our findings provide further evidence for the functional specialization of different CD1 isoforms and demonstrate the value of the fluorescent lipid probe binding method for assisting structure-based studies of CD1 function.     

Specific isoforms of the resident endoplasmic reticulum protein glucosidase II associate with the CD45 protein-tyrosine phosphatase via a lectin-like interaction

We have previously demonstrated that CD45 physically associates with the endoplasmic reticulum processing enzyme glucosidase II (GII). GII consists of the catalytic alpha-chain and an associated beta-chain. To gain insight into the basis of the association between CD45 and GII, we examined the biochemical requirements for the interaction. We show that the alpha-subunit is essential for the interaction. Interestingly, only a higher molecular weight form of GIIalpha is capable of associating with CD45 in a competitive situation where multiple GIIalpha isoforms are expressed. Further, transfection studies demonstrate that only isoforms containing the alternatively spliced sequence Box A1 are capable of binding CD45, although all isoforms are catalytically active. The interaction between CD45 and GII is dependent on the active site of GII, is mediated through the carbohydrate on CD45, and can be inhibited with mannose. Taken together, these results suggest that GIIalpha acts as a lectin and binds to CD45 in an exon-dependent manner. This lectin activity of GII may be a novel mechanism for the regulation of CD45 biology and play a role in immune function, possibly by regulating CD45 glycosylation.     

PECAM-1/CD31 trans-homophilic binding at the intercellular junctions is independent of its cytoplasmic domain; evidence for heterophilic interaction with integrin alphavbeta3 in Cis

PECAM-1/CD31 is a cell adhesion and signaling molecule that is enriched at the endothelial cell junctions. Alternative splicing generates multiple PECAM-1 splice variants, which differ in their cytoplasmic domains. It has been suggested that the extracellular ligand-binding property, homophilic versus heterophilic, of these isoforms is controlled by their cytoplasmic tails. To determine whether the cytoplasmic domains also regulate the cell surface distribution of PECAM-1 splice variants, we examined the distribution of CD31-EGFPs (PECAM-1 isoforms tagged with the enhanced green fluorescent protein) in living Chinese hamster ovary cells and in PECAM-1-deficient endothelial cells. Our results indicate that the extracellular, rather than the cytoplasmic domain, directs PECAM-1 to the cell-cell borders. Furthermore, coculturing PECAM-1 expressing and deficient cells along with transfection of CD31-EGFP cDNAs into PECAM-1 deficient cells reveal that this PECAM-1 localization is mediated by homophilic interactions. Although the integrin alphavbeta3 has been shown to interact with PECAM-1, this trans-heterophilic interaction was not detected at the borders of endothelial cells. However, based on cocapping experiments performed on proT cells, we provide evidence that the integrin alphavbeta3 associates with PECAM-1 on the same cell surface as in a cis manner.     

An essential role of sialylated O-linked sugar chains in the recognition of mouse CD99 by paired Ig-like type 2 receptor (PILR)

The paired Ig-like type 2 receptor (PILR), which comprises both inhibitory and activating isoforms, is well conserved among most mammalians. The inhibitory PILRalpha possesses an ITIM in its cytoplasmic domain, whereas the activating PILRbeta does not have an ITIM but transduces activating signals by associating with the ITAM-bearing DAP12 adapter molecule. Both mouse PILRalpha and PILRbeta recognize mouse CD99, which is broadly expressed on various cells, including lymphocytes, and is involved in the regulation of immune responses. We herein report that sialylated O-linked sugar chains on CD99 are essential for the recognition by PILR. Mutations of one of two O-glycosylation sites on CD99 significantly reduced recognition of CD99 by the activating PILRbeta, whereas recognition by the inhibitory PILRalpha was not affected. In contrast, mutations of both O-glycosylation sites on CD99 completely abrogated the recognition by both PILRalpha and PILRbeta. PILR did not recognize CD99 treated with neuraminidase, and CD99 expressed on cells transfected with core 2 beta-1,6-N-acetylglucosaminyltransferase was not recognized by PILR. NK cells expressing endogenous activating PILRbeta receptors mediated cytotoxicity against cells expressing wild-type CD99 but not cells expressing mutant CD99 that lacked O-glycosylation sites. These findings indicate that sialylated O-linked sugar structures on CD99 play an important role in the recognition of PILR.     

Loss of N-terminal charged residues of mouse CD3 epsilon chains generates isoforms modulating antigen T cell receptor-mediated signals and T cell receptor-CD3 interactions

The antigen T cell receptor (TCR)-CD3 complexes present on the cell surface of CD4(+) T lymphocytes and T cell lines express CD3 epsilon chain isoforms with different isoelectric points (pI), with important structural and functional consequences. The pI values of the isoforms fit the predicted pI values of CD3 epsilon chains lacking one, two, and three negatively charged amino acid residues present in the N-terminal region. Different T cells have different ratios of CD3 epsilon chain isoforms. At a high pI, degraded CD3 epsilon isoforms can be better recognized by certain anti-CD3 monoclonal antibodies such as YCD3-1, the ability of which to bind to the TCR-CD3 complex is directly correlated with the pI of CD3 epsilon. The abundance of CD3 epsilon isoforms can be modified by treatment of T cells with the proteinase inhibitor phenanthroline. In addition, these CD3 epsilon isoforms have functional importance. This is shown, first, by the different structure of TCR-CD3 complexes in cells possessing different amounts of isoforms (as observed in surface biotinylation experiments), by their different antigen responses, and by the stronger interaction between low pI CD3 epsilon isoforms and the TCR. Second, incubation of cells with phenanthroline diminished the proportion of degraded high pI CD3 epsilon isoforms, but also the ability of the cells to deliver early TCR activation signals. Third, cells expressing mutant CD3 epsilon chains lacking N-terminal acid residues showed facilitated recognition by antibody YCD3-1 and enhanced TCR-mediated activation. Furthermore, the binding avidity of antibody YCD3-1 was different in distinct thymus populations. These results suggest that changes in CD3 epsilon N-terminal chains might help to fine-tune the response of the TCR to its ligands in distinct activation situations or in thymus selection.     

Effect of bridging the two essential thiols of myosin on its spectral and actin-binding properties.

The circular dichroic and fluorescent spectral properties of the myosin head (subfragment I (SFI)) modified by covalently bridging the two essential thiol groups have been examined. CD spectra of SFI with the two thiols linked through reaction with a bifunctional reagent, N, N'- p-phenylenedimaleimide, show enhancement of the 282-nm minimum similar to that observed for the long-lived kinetic intermediate (Mg**MgADP-Pi) formed during the ATP cleavage reaction. No significant perturbation of the CD band at 282 nm is seen on blocking both thiol groups with the monofunctional reagent N-ethylmaleimide. The fluorescence emission maximum also shifts to lower wavelengths following covalent bridging (from 343 to 340 nm), but no change in fluorescent intensity has been detected. Formation of the covalent bridge completely inhibits interaction of the modified protein with F-actin. These results suggest that the local conformational state of the polypeptide chain formed on bridging the two thiol groups exhibits certain similarities with the state produced following binding of MgATP to native myosin.     

The secondary structure of turkey gizzard myosin light chain kinase and the nature of its interaction with calmodulin

The enzymatic activities of native myosin light chain kinases are subject to modification by interaction with Ca2(+)-calmodulin (CaM). The interaction between myosin light chain kinase isolated from turkey gizzard (tgMLCK) and calmodulin isolated from bovine testes (CaMbt) and wheat germ (CaMwg) has been examined by means of the intrinsic tryptophan fluorescence of tgMLCK and the fluorescence of extrinsic fluorescent labels located at Cys-27 and Tyr-139 of CaMwg and Tyr-99 of CaMbt. Static and dynamic fluorescence measurements provide evidence for the involvement of the former two sites in the zone of contact with lesser involvement of the site marked by the probe at Tyr-99. Complex formation protected the primary cleavage site in CaMbt (Lys-77) from proteolysis by trypsin. These results are consistent with involvement of the N- and C-terminal lobes of CaM in stabilization of the complex with tgMLCK, but cannot rule out participation of the connecting strand in the interaction. CD measurements extending to 175 nm, obtained using synchroton radiation, indicate the following secondary structure content for tgMLCK: 17 +/- 2% alpha-helix, 22 +/- 3% antiparallel beta-sheet, 3 +/- 1% parallel beta-sheet, 24 +/- 2% beta-turns, and 34 +/- 2% random coil. Similar measurements of the CD spectra of CaMbt and of the 1:1::CaMbt:tgMLCK complex presently indicate that neither protein undergoes major secondary structure rearrangement during their interaction, although subtle changes in the CD spectrum of tgMLCK appear to be correlated with the interaction with CaM.     

CD99 is a key mediator of the transendothelial migration of neutrophils

Transendothelial migration of leukocytes is a critical event for inflammation, but the molecular regulation of this event is only beginning to be understood. PECAM (CD31) is a major mediator of monocyte and neutrophil transmigration, and CD99 was recently defined as a second mediator of the transmigration of monocytes. Expression of CD99 on the surface of circulating polymorphonuclear cells (PMN) is low compared with expression of CD99 on monocytes or expression of PECAM on PMN. We demonstrate here that, despite low expression of CD99, Fab of Abs against CD99 blocked over 80% of human neutrophils from transmigrating across HUVEC monolayers in an in vitro model of inflammation. Blocking CD99 on either the neutrophil or endothelial cell side resulted in a quantitatively equivalent block, suggesting a homophilic interaction between CD99 on the neutrophil and CD99 on the endothelial cell. Blocking CD99 and PECAM together resulted in additive effects, suggesting the two molecules work at distinct steps. Confocal microscopy confirmed that CD99-blocked neutrophils lodged in endothelial cell junctions at locations distal to PECAM-blocked neutrophils. The CD99-blocked PMN exhibited dynamic lateral movement within endothelial cell junctions, indicating that only the diapedesis step was blocked by interference with CD99. Anti-CD99 mAb also blocked PMN transmigration in a second in vitro model that incorporated shear stress. Taken together, the evidence demonstrates that PECAM and CD99 regulate distinct, sequential steps in the transendothelial migration of neutrophils during inflammation.     

CD44 is not an adhesive receptor for osteopontin

Osteopontin is a secreted glycoprotein with adhesive and migratory functions. Cellular interactions with osteopontin are mediated through integrin receptors which recognize the RGD domain. Recently, CD44, a non-integrin, multifunctional adhesion molecule was identified as an osteopontin receptor. CD44 is a ubiquitous surface molecule that exists as a number of different isoforms, generated by alternative splicing. To analyze which forms of CD44 mediate binding to osteopontin, we used the standard form of CD44 as CD44-human immunoglobulin fusion proteins and several splice variants in enzyme-linked immunosorbant assays. Multiple preparations of osteopontin were used including native osteopontin derived from smooth muscle cells, human urinary osteopontin, full-length recombinant osteopontin, and two recombinant osteopontin fragments expected to be formed following thrombin cleavage. Our data show that although the CD44-hlg fusion proteins could interact with hyaluronic acid as expected, there was no interaction between CD44H, CD44E, CD44v3,v8-v10, or CD44v3 with osteopontin. These studies suggest that CD44-osteopontin interactions may not be common in vivo and may be limited to a specific CD44 isoform(s), and/or a particular modified form of osteopontin.     

两种存活蛋白变异剪接体在HeLa细胞中的表达定位及相互作用

存活蛋白(survivin)是重要的肿瘤相关抗原基因,在肿瘤的发生发展中 起着重要的作用. 除了标准的剪接形式外,它至少还编码2种变异剪接产物—存活蛋白-2B 和存活蛋白-ΔEx3,这2个变异剪接体所编码的蛋白具有不同的生物学功能.为研究这2个变 异剪接体在肿瘤细胞中的相互作用情况,本实验利用增强型青色荧光蛋白（enhanced cyan fluorescent protein, ECFP）和增强型黄色荧光蛋白（enhanced yellow fluorescent protein, EYFP）分别标记存活蛋白-2B 和存活蛋白-ΔEx3.首先通过激光共聚焦扫描显微镜观 察它们的细胞定位;同时利用荧光共振能量转移（fluorescence resonance energy transf er, FRET）技术研究两者在细胞内的相互作用情况.研究结果表明, 存活蛋白-2B主要分布 在细胞质中,而存活蛋白-ΔEx3则主要分布在细胞核内,少量分布在细胞质中;FRET分析结 果显示,两者仅在细胞质中存在着很弱的相互作用,表明两者很可能是通过某种间接的方式发 挥功能上的相互调节作用.本研究为进一步探讨存活蛋白变异剪接体的生物学功能及相互作用 机制奠定了基础.     

CD99 is essential for leukocyte diapedesis in vivo

Recruitment of leukocytes into inflamed tissue requires migration of leukocytes from the blood stream across the endothelial lining and the basement membrane of the local blood vessels. CD99 in humans is a 32-kDa highly O-glycosylated cell surface protein expressed on most leukocytes. The authors recently found CD99 to be expressed in leukocytes and at human endothelial cell contacts. Human CD99 is involved in homophilic interaction between the two cell types and participates in the transendothelial migration of monocytes and polymorphonuclear neutrophils (PMNs) in vitro. To test the role of CD99 in vivo, the authors cloned murine CD99 (muCD99), expressed it in vitro, and generated a blocking monoclonal antibody against it. We first showed that muCD99 is expressed on mouse leukocytes as well as enriched at the endothelial cell borders. Transfection of cells with muCD99 imparts on them the ability to aggregate in a CD99-dependent homophilic manner. Cells expressing muCD99 did not bind to cells expressing murine or human platelet endothelial call adhesion molecule (PECAM) or human CD99. In the thioglycollate peritonitis model of inflammation, anti-CD99 monoclonal antibody blocked the recruitment of neutrophils and monocytes by over 40% and 80%, respectively, at 18 h. Microscopy showed that this blocking occurred at the luminal surface of venules. The authors conclude that CD99 plays a major role in the emigration of leukocytes in vivo.     

Molecular organization, structural features, and ligand binding characteristics of CD44, a highly variable cell surface glycoprotein with multiple functions

CD44 is a type I transmembrane protein and member of the cartilage link protein family. It is involved in cell-cell and cell-matrix interactions and signal transduction. Several CD44 ligands have been identified. CD44 is a major cell surface receptor for hyaluronan, a component of the extracellular matrix. It is implicated in diseases such as cancer and inflammation and therefore intensely studied. A characteristic feature of CD44 is the occurrence of many isoforms that are expressed in a cell-specific manner and differentially glycosylated. Although a number of CD44 isoforms have been characterized, the structural diversity of CD44 makes it often challenging to study (isoform-specific) CD44-ligand interactions at the molecular level of detail. The structural organization and ligand binding characteristics of CD44 are focal points of this review. On the basis of recent structural and mutagenesis studies, details of the CD44-hyaluronan interaction are beginning to be understood. Proteins 2000;39:103-111.     

Competition for microtubule-binding with dual expression of tau missense and splice isoforms

How tau mutations lead to neurodegeneration is unknown but may be related to altered microtubule binding properties of mutant tau protein. The tendency for the mutations to cluster around the microtubule-binding domain of tau or to alter the ratios of those splice isoforms that affect binding supports the view that the tau/microtubule interaction is critical and finely regulated. In cells transfected with both mutant and wild-type tau isoforms fused to either yellow fluorescent protein or cyan fluorescent protein we can observe tau fusion proteins that differ by a single amino acid or by the inclusion or exclusion of exon 10. With coexpression of mutant and wild-type tau, the mutant isoform appears diffuse throughout the cytoplasm; however, when mutant tau is expressed alone, it appears mostly bound to the microtubules. Dual imaging of the three- and four-repeat tau isoforms indicated that the expression of four-repeat tau displaced three-repeat tau from the microtubules. These results suggest that altered kinetic competition among the isoforms for microtubule binding could be a disease precipitant.     

Distinct Kinetic and Molecular Requirements Govern CD44 Binding to Hyaluronan versus Fibrin(ogen)

CD44 is a multifunctional glycoprotein that binds to hyaluronan and fibrin(ogen). Alternative splicing is responsible for the generation of numerous different isoforms, the smallest of which is CD44s. Insertion of variant exons into the extracellular membrane proximal region generates the variant isoforms (CD44v). Here, we used force spectroscopy to delineate the biophysical and molecular requirements of CD44-HA and CD44-fibrin(ogen) interactions at the single-molecule level. CD44v-HA and CD44s-HA single bonds exhibit similar kinetic and micromechanical properties because the HA-binding motif on CD44 is common to all of the isoforms. Although this is the primary binding site, O- and N-linked glycans and sulfation also contribute to the tensile strength of the CD44-HA bond. The CD44s-fibrin pair has a lower unstressed dissociation rate and a higher tensile strength than CD44s-fibrinogen but is weaker than the CD44-HA bond. In contrast to CD44-HA binding, the molecular interaction between CD44 and fibrin(ogen) is predominantly mediated by the chondroitin sulfate and dermatan sulfate on CD44. Blocking sulfation on CD44s modestly decreases the tensile strength of CD44s-fibrin(ogen) binding, which is in stark contrast to CD44v-fibrin interaction. Collectively, the results obtained by force spectroscopy in conjunction with biochemical interventions enable us to delineate the biophysical parameters and molecular constituents of CD44 binding to hyaluronan and fibrin(ogen).     

CD44 in rheumatoid arthritis

CD44 is a multistructural cell-surface glycoprotein that can theoretically generate close to 800 isoforms by differential alternative splicing. At present, several dozen isoforms are known. The polymorphic nature of CD44 might explain its multifunctionality and its ability to interact with many cell-surface and extracellular ligands, the principal one being hyaluronic acid (HA). Of the many CD44 functions, our review focuses on its involvement in cell–cell and cell–matrix interactions, as well as on its implication in the support of cell migration and the presentation of growth factors to their cognate receptors. Cells involved in pathological activities such as cancer cells and destructive inflammatory cells, and also normal cells engaged in physiological functions, use cell-surface CD44 for their localization and expansion at extravascular sites. This article reviews the evidence that the joint synovium of patients with rheumatoid arthritis (RA) contains considerable amounts of various CD44 isoforms as well as the HA ligand. The review also shows that anti-CD44 monoclonal antibody (mAb) directed against constant epitopes, shared by all CD44 isoforms, can markedly reduce the inflammatory activity of arthritis induced by collagen or proteoglycans in mice. Anti-CD44 mAb also interferes with the migration of RA synovial-like fibroblasts in vitro and is able to disturb the destructive interaction between RA synovial-like fibroblasts and the cartilaginous matrix. However, the transition from the experimental model to the patient's bedside is dependent on the ability to target the CD44 of cells engaged in RA pathology, while skipping the CD44 of normal cells.     

Identification of sequence motifs responsible for the adhesive interaction between exon v10-containing CD44 isoforms

Previous studies have demonstrated that CD44 isoforms containing the alternatively spliced exon v10 promote cell-cell adhesion via a mechanism that involves the recognition of chondroitin sulfate side chains presented on the surface of interacting cells in association with other CD44 molecules. Sequence analysis revealed the presence within exon v10 of two motifs that may be relevant to this interaction, a B[X(7)]B motif that may contribute to the recognition and binding of chondroitin sulfate and a serine-glycine motif that may serve as a site of chondroitin sulfate attachment. To determine whether either of these two motifs explain the unique adhesive activity of exon v10-containing CD44 isoforms, each was targeted by site-directed mutagenesis, and the adhesive activity of the resultant mutants was determined using a quantitative cell-cell binding assay. The data obtained demonstrate conclusively that it is the exon v10-encoded B[X(7)]B motif that is solely responsible for the enhanced adhesive activity of exon v10-containing CD44 isoforms.     

Dynamic assembly properties of nonmuscle myosin II isoforms revealed by combination of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

Myosin II molecules assemble into filaments through their C-terminal rod region, and are responsible for several cellular motile activities. Three isoforms of nonmuscle myosin II (IIA, IIB and IIC) are expressed in mammalian cells. However, little is known regarding the isoform composition in filaments. To obtain new insight into the assembly properties of myosin II isoforms, especially regarding the isoform composition in filaments, we performed a combination analysis of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS), which enables us to acquire information on both the interaction and the size of each molecule simultaneously. Using C-terminal rod fragments of IIA and IIB (ARF296 and BRF305) labelled with different fluorescent probes, we demonstrated that hetero-assemblies were formed from a mixture of ARF296 and BRF305, and that dynamic exchange of rod fragments occurred between preformed homo-assemblies of each isoform in an isoform-independent manner. We also showed that Mts1 (S100A4) specifically stripped ARF296 away from the hetero-assemblies, and consequently, homo-assemblies of BRF305 were formed. These results suggest that IIA and IIB can form hetero-filaments in an isoform-independent manner, and that a factor like Mts1 can remove one isoform from the hetero-filament, resulting in a formation of homo-filaments consisting of another isoform.