T cell receptor/CD3 negative variants are unresponsive to stimulation through the Ly-6 encoded molecule, TAP

TAP is a phosphatidylinositol-anchored Ly-6-encoded, glycoprotein that is involved in the activation of murine T-inducer cells. Anti-TAP antibodies can stimulate T cells directly and also synergize with Ag stimulation. To determine the relationship between TAP and TCR-mediated activation, we have derived TCR/CD3 loss variants of functional T hybrid clones. T-T hybrid variants that have lost TCR expression are not responsive to anti-TAP stimulation. Isolation and analysis of revertant clones, obtained from one of the TCR/CD3-negative mutant cell lines, demonstrate a concordant re-expression of the TCR/CD3 complex and responsiveness to anti-TAP stimulation. A similar loss of responsiveness to TAP stimulation is observed after antibody-induced modulation of the TCR/CD3 complex from the surface of T cell hybrids. In contrast, both the TCR/CD3 loss variants and TCR/CD3-modulated T-T hybrids remain fully responsive to stimulation with a calcium ionophore and phorbol esters. This structure-function correlation has been observed repeatedly in independent isolations of variants, and with TCR/CD3 modulated cells, from two different T-T hybridomas. Given the apparent functional interrelationship between TAP and the TCR/CD3 complex, we have also analyzed if these molecules were significantly associated on the T cell surface. Antibody-induced modulation of the TCR/CD3 complex does not affect the cell surface expression of TAP molecules. Moreover, the expression of these two sets of proteins is also independent as evidenced by the selective loss of either set of these proteins in the TCR/CD3 expression mutants.     

Ly-6I, a new member of the murine Ly-6 superfamily with a distinct pattern of expression

A new member of the mouse Ly-6SF, designated Ly-6I, has been isolated as a gene homologous to a segment of the Ly-6C gene. A single allelic difference in the mature protein sequence was identified, which is similar to other Ly-6SF members. Ly-6I mRNA has been detected in a wide range of tissues and cell lines, and a rabbit polyclonal Ab has been used to determine that Ly-6I protein is present at a low constitutive level on cell lines from several different lineages. In contrast to Ly-6C and Ly-6A/E, the Ly-6I gene is only weakly responsive to IFNs. Expression in vivo is most abundant on bone marrow populations and is coexpressed with Ly-6C on granulocytes and macrophages. However, Ly-6I is also expressed on immature B cell populations that do not express Ly-6C. Expression on mature B cells in spleen is uniformly low. Similarly, Ly-6I is expressed on TCRlow/int, but not TCRhigh, thymocytes. Ly-6I is re-expressed on Ly-6Chigh T cells in the periphery. Thus, Ly-6I may be a useful marker to define maturation stages of both T and B lymphocytes as well as subsets of monocytes and granulocytes.     

Stimulation of murine T cells via the Ly-6C antigen: lack of proliferative response in aberrant T cells from lpr/lpr and gld/gld mice despite high Ly-6C antigen expression

Cross-linking of cell surface Ly-6C molecules with the 6C3 rat monoclonal antibody (MAb) followed by anti-rat immunoglobulin antibody acts in concert with phorbol myristate acetate (PMA) as a potent mitogenic stimulus for normal T cells. Specificity of this stimulation was demonstrated by its absence in T cells from NZB, NOD, or STb/J mice which lack the 6C3 determinant. In 6C3+ normal strains, the extent of 6C3-mediated stimulation varied, depending on the level of 6C3 antigen expression. Analysis of this stimulation in purified T cell subsets revealed that in Ly-6.1 strains (e.g., BALB/c, CBA/J), Lyt-2+ cells responded, but not L3T4+ cells, whereas in Ly-6.2 strains (e.g., C57BL/6, MRL-+/+), both subsets produced IL 2 and proliferated, although with different kinetics. Moreover, in adult MRL-+/+ mice, the minor Lyt-2-/L3T4- subset from the lymph nodes gave low responses to 6C3 cross-linking, whereas that from the thymus reacted strongly. Stimulation via Ly-6C therefore provides a pathway for differential activation of normal T cells. In contrast, the expanding population of Lyt-2-/L3T4- T cells from lpr/lpr or gld/gld mice did not proliferate in response to 6C3 antigen cross-linking plus PMA despite high levels of 6C3 antigen expression. Responsiveness of lpr/lpr T cells could not be restored with IL 1, IL 2, or both. These T cells also failed to be triggered by conjunction of PMA with either Thy-1 antigen cross-linking or concanavalin A. Moreover, they were not stimulated, in the presence of PMA, by doses of ionomycin that were optimal for normal T cells, but did respond to higher ionomycin concentrations (2 micrograms/ml), and this response was not altered by Ly-6C cross-linking. It is concluded that the Ly-6C pathway of T cell activation is not functional in the aberrant lpr/lpr (and gld/gld) T cells, and that this defect may reflect abnormalities of intracellular signaling.     

A human T cell-specific molecule is a member of a new gene family

We have used a cDNA library enriched for T cell-specific sequences to isolate genes expressed by T cells but not by other cell types. We report here one such gene, designated RANTES, which encodes a novel T cell-specific molecule. The RANTES gene product is predicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines, and there are no sites for N-linked glycosylation. RANTES is expressed by cultured T cell lines that are Ag specific and growth factor dependent. RANTES expression is inducible in PBL by Ag or mitogen. In CTL, expression of RANTES decreases after stimulation with Ag and growth factors. Interestingly, RANTES was not expressed by any T cell tumor line tested. There is significant homology between the RANTES sequence and several other T cell genes, suggesting that they comprise a previously undescribed family of small T cell molecules.     

Definition of new alloantigens encoded by genes in the Ly-6 complex

Three alloantigens encoded by Ly-6-linked genes are defined by monoclonal antibodies. The Ly-27.2 antigen is defined by antibody 5075-19.1, Ly-28.2 by 5075-3.6, -12.1, -16.10 and by 5095-16.6. The strain distribution pattern of these antibodies is the same and identical with Ly-6.2. However the tissue distribution of these antigens is unique and distinguishes these antigens from the Ly-6.2 antigen or any known antigen encoded by Ly-6-linked genes. Ly-27.2 is present on all thymocytes, T cells, and B cells but is absent from bone marrow cells, whereas Ly-28.2 is absent from most thymocytes and is present on a subpopulation of T cells and B cells but is found on 60–70% of bone marrow cells. No recombination between the Ly-6/Ly-27/Ly-28 loci was found in linkage studies using 41 recombinant inbred strains and 57 backcross mice and indicates very close linkage of these genes. In addition, close linkage to 24 minor histocompatibility genes was excluded using the Bailey HW bilineal congenic mice. The data presented indicate that either the Ly-6 complex is composed of a family of tightly linked genes or the antigens are the products of a single gene that undergoes extensive modification during differentiation.     

CELF6, a member of the CELF family of RNA-binding proteins, regulates muscle-specific splicing enhancer-dependent alternative splicing

We previously described a family of five RNA-binding proteins: CUG-binding protein, embryonic lethal abnormal vision-type RNA-binding protein 3, and the CUG-binding protein and embryonic lethal abnormal vision-type RNA-binding protein 3-like factors (CELFs) 3, 4, and 5. We demonstrated that all five of these proteins specifically activate exon inclusion of cardiac troponin T minigenes in vivo via muscle-specific splicing enhancer (MSE) sequences. We also predicted that a sixth family member, CELF6, was located on chromosome 15. Here, we describe the isolation and characterization of CELF6. Like the previously described CELF proteins, CELF6 shares a domain structure containing three RNA-binding domains and a divergent domain of unknown function. CELF6 is strongly expressed in kidney, brain, and testis and is expressed at very low levels in most other tissues. In the brain, expression is widespread and maintained from the fetus to the adult. CELF6 activates exon inclusion of a cardiac troponin T minigene in transient transfection assays in an MSE-dependent manner and can activate inclusion via multiple copies of a single element, MSE2. These results place CELF6 in a functional subfamily of CELF proteins that includes CELFs 3, 4, and 5. CELF6 also promotes skipping of exon 11 of insulin receptor, a known target of CELF activity that is expressed in kidney.     

Biochemical characterization of the murine activated lymphocyte alloantigen Ly-6E.1 controlled by the Ly-6 locus

The Ly-6.1 alloantigen defined by monoclonal antibody SK70.94 and expressed predominantly on activated lymphocytes has been immunoprecipitated from lysates of cells biosynthetically radiolabeled with 35S-methionine. On SDS-polyacrylamide gel electrophoresis the reduced antigen appeared as a doublet of 17 and 18 kD. The nonreduced polypeptides had higher mobilities indicating intrachain but not interchain disulfide linkages. The nonreduced form was detectable by immunoperoxidase stain on blots after SDS-PAGE. This showed both polypeptide species contained the antigenic epitope. Labeling in the presence of tunicamycin did not change the apparent m.w. suggesting the absence of N-linked carbohydrate. Pulse-chase data are inconsistent with a strict precursor-product relationship between the two polypeptides and give a half-life for the antigen in 2-day Con A blast cells of about 4 1/2 hr. A highly purified preparation of this antigen displayed a similar electrophoretic pattern and, in the presence of deoxycholate, eluted from a Sephacryl S-200 gel filtration column with a partition coefficient equivalent to that of a 31-kD soluble globular protein. Because of the associated detergent and probable deviation from globularity, this is an over-estimate of the size of the native molecule, and is more consistent with the native molecule being a single polypeptide chain rather than a dimer. Isoelectric focusing of this material showed microheterogeneity with at least five bands between pI 4 and 5. Another monoclonal antibody, HD-42, which had been isolated on the basis of its specificity for a fibrosarcoma antigen subsequently found to be Ly-6 related, precipitated the same polypeptides. Furthermore, no obvious difference was evident between precipitates from Con A-activated lymphocytes and Meth A fibrosarcoma cells.     

Drosophila chaoptin, a member of the leucine-rich repeat family, is a photoreceptor cell-specific adhesion molecule

Drosophila chaoptin, required for photoreceptor cell morphogenesis, is a member of the leucine-rich repeat family of proteins. On the basis of biochemical and genetic analyses we previously proposed that chaoptin might function as a cell adhesion molecule. To test this hypothesis, chaoptin cDNA driven by the hsp 70 promoter was transfected into non-self-adherent Drosophila Schneider line 2 (S2) cells. Following heat shock induction of chaoptin expression, the transfected S2 cells formed multicellular aggregates. Mixing experiments of chaoptin expressing and non-expressing cells suggest that chaoptin expressing cells adhere homotypically. Previously it was shown that chaoptin is exclusively localized to photoreceptor cells. Thus, chaoptin is a cell-type-specific adhesion molecule. Biochemical analyses presented in this paper demonstrate that chaoptin is linked to the extracellular surface of the plasma membrane by covalent attachment to glycosyl-phosphatidylinositol. We propose that chaoptin and several other members of the leucine-rich repeat family of proteins define a new class of cell adhesion molecules.     

Characterization of the five novel Ly-6 superfamily members encoded in the MHC, and detection of cells expressing their potential ligands

Lymphocyte Antigen 6 (Ly-6) superfamily members are cysteine-rich, generally GPI-anchored cell surface proteins, which have definite or putative immune related roles. There are 27 members of this family described so far in the human genome and 37 in the mouse. Five of them are clustered in the class III region of the human and mouse MHCs. Following computational analyses, we functionally characterized the encoded proteins by creating epitope-tagged fusion constructs to determine molecular weight, complex formation, subcellular localization, post-translational modifications and ligand binding. We found that all human and mouse proteins were glycosylated, and most could form part of larger complexes. Human and mouse Ly6G6c and Ly6G6d, and mouse Ly6g6e were found to be GPI-anchored cell surface proteins, highly expressed at the leading edges of cells, on filopodia, which are normally involved in cell adhesion and migration. However, analysis of Ly6G5c and Ly6G5b indicated that they are potentially secreted proteins. Our results indicate that there are two subclusters of related Ly-6 proteins in this region of the MHC, with Ly6G6c, Ly6G6d, and Ly6G6e forming one and Ly6G5c and Ly6G5b forming another. In addition, by FACS analysis we have found that the potential ligands for human LY6G6C, LY6G6D, and LY6G5C are expressed on K562 cells, an undifferentiated megakaryocyte cell line, indicating a potential role in hematopoietic cell differentiation. This characterization of the five MHC class III region Ly-6 family members is of great relevance, as they represent 18% of the human Ly-6 protein family and 50% of the secreted ones.     

T cell activation via CD2 [T, gp50]: the role of accessory cells in activating resting T cells via CD2

Monoclonal antibody (MAb) GT2 defines a unique epitope on the CD2 molecule. GT2 triggers T cell mitosis in combination with any MAb directed against 9.6/T11(1) or D66, two previously defined CD2 epitopes. We have shown already that accessory cells (AC) are required for plenary T-PBL activation by any pair of Ab directed against D66 + 9.6/T11(1). In this study, we further investigated their role and found it to vary with the anti-CD2 pair used. When purified T-PBL preparation is used, the level of [3H]TdR incorporation observed with anti-(GT2 + 9.6/T11(1)) Ab was not significant; however, it did prove significant, although greatly reduced, with the other anti-CD2 pairs tested. This was due to qualitative differences in the process of T-PBL activation, and the role of AC, because: anti-(GT2 + 9.6/T11(1)) did not induce IL 2-R expression on purified T-PBL, whereas the other anti-CD2 pairs tested did; anti-(GT2 + 9.6/T11(1)) did not induce detectable IL 2 secretion from purified T-PBL, whereas the other anti-CD2 pairs tested induced a low amount; and anti-CDw18 Ab inhibited the mitogenic effect of anti-(GT2 + 9.6/T11(1)) on PBMC by preventing both IL 2-R expression and IL 2 secretion, whereas anti-CDw18 Ab enhanced the mitogenic effect of the other anti-CD2 pairs tested. Paraformaldehyde-fixed AC fully restored, and recombinant IL 1 partially restored purified T-PBL mitosis triggered by all anti-CD2 pairs tested. To induce IL 2 synthesis, the necessity to cross-link anti-CD2 Ab was demonstrated by coupling one Ab on Sepharose beads and adding the second Ab in the soluble phase: under these circumstances, anti-CD2 pairs were mitogenic solely in the presence of AC. These data can be interpreted as follows. Most anti-CD2 pairs require minimal contact between AC and T-PBL to induce plenary levels of IL 2 synthesis. When anti-(GT2 + 9.6/T11(1)) are used, additional contact is necessary, both for IL 2-R expression and IL 2 synthesis, which would include CDw18 for stabilization. We believe these differences could be related to different conformational changes on the CD2 molecule, depending on the epitope on which the antibodies bind, and could account for different signaling to T cells.     

Plant cells contain a novel member of the retinoblastoma family of growth regulatory proteins.

The product of the retinoblastoma susceptibility gene (Rb) controls the passage of mammalian cells through G1 phase. Animal virus oncoproteins interact with the Rb protein via an LXCXE motif and disrupt Rb-E2F complexes, driving cells into S-phase. Recently, we found that the RepA protein of a plant geminivirus contains an LXCXE motif that is essential for its function, a finding that predicts the existence of Rb-related proteins in plant cells. Here we report the isolation of a maize cDNA clone encoding a protein (ZmRb1) which, based on structural and functional studies, is closely related to the mammalian Rb family of growth regulatory proteins. ZmRb1 shows a high degree of amino acid conservation when compared with animal Rb members, particularly in the A/B 'pocket' domain, but ZmRb1 has a shorter N-terminal domain. ZmRb1 forms stable complexes with plant LXCXE-containing proteins, e.g. geminivirus RepA protein. Geminivirus DNA replication is reduced in plant cells transfected with plasmids encoding either ZmRb1 or human p130, a member of the Rb family. This suggests that ZmRb1 controls the G1/S transit in plant cells and is consistent with the fact that geminiviruses need an S-phase environment for DNA replication, as animal DNA tumor viruses do. Our results allow the extension of the Rb family of tumor suppressor proteins to plants and have implications on animal and plant strategies for cell growth control.     

Periostin, a member of a novel family of vitamin K-dependent proteins, is expressed by mesenchymal stromal cells

The modification of glutamic acid residues to gamma-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent enzyme gamma-glutamylcarboxylase. Despite ubiquitous expression of the gamma-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-containing proteins after the liver, we sought to identify Gla proteins secreted by bone marrow-derived mesenchymal stromal cells (MSCs). We used a proteomics approach to screen the secretome of MSCs with a combination of two-dimensional gel electrophoresis and tandem mass spectrometry. The most abundant Gla-containing protein secreted by MSCs was identified as periostin, a previously unrecognized gamma-carboxylated protein. In silico amino acid sequence analysis of periostin demonstrated the presence of four consensus gamma-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation and purification of the recombinant protein. Carboxylation of periostin could be inhibited by warfarin in MSCs, demonstrating its dependence on the presence of vitamin K. We were able to demonstrate localization of carboxylated periostin to bone nodules formed by MSCs in vitro, suggesting a role in extracellular matrix mineralization. Our data also show that another fasciclin I-like protein, betaig-h3, contains Gla. In conclusion, periostin is a member of a novel vitamin K-dependent gamma-carboxylated protein family characterized by the presence of fasciclin domains. Furthermore, carboxylated periostin is produced by bone-derived cells of mesenchymal lineage and is abundantly found in mineralized bone nodules in vitro.     

Cellular and subcellular localization of EFA6C, a third member of the EFA6 family, in adult mouse Purkinje cells

EFA6C is a third member of the EFA6 family of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factor 6 (ARF6). In this study, we first demonstrated that EFA6C indeed activated ARF6 more selectively than ARF1 by ARF pull-down assay. In situ hybridization histochemistry revealed that EFA6C mRNA was expressed predominantly in mature Purkinje cells and the epithelial cells of the choroid plexus in contrast to the ubiquitous expression of ARF6 mRNA throughout the brain. EFA6C mRNA was already detectable in the Purkinje cells at embryonic day 13, increased progressively during post-natal development and peaked during post-natal second week. In Purkinje cells, the immunoreactivity for EFA6C was localized particularly in the post-synaptic density as well as the plasma membranes of the cell somata, dendritic shafts and spines, while the immunoreactivity in their axon terminals in the deep cerebellar nuclei was very faint. These findings suggest that EFA6C may be involved in the regulation of the membrane dynamics of the somatodendritic compartments of Purkinje cells through the activation of ARF6.     

The sodium-activated potassium channel is encoded by a member of the Slo gene family

Na(+)-activated potassium channels (K(Na)) have been identified in cardiomyocytes and neurons where they may provide protection against ischemia. We now report that K(Na) is encoded by the rSlo2 gene (also called Slack), the mammalian ortholog of slo-2 in C. elegans. rSlo2, heterologously expressed, shares many properties of native K(Na) including activation by intracellular Na(+), high conductance, and prominent subconductance states. In addition to activation by Na(+), we report that rSLO-2 channels are cooperatively activated by intracellular Cl(-), similar to C. elegans SLO-2 channels. Since intracellular Na(+) and Cl(-) both rise in oxygen-deprived cells, coactivation may more effectively trigger the activity of rSLO-2 channels in ischemia. In C. elegans, mutational and physiological analysis revealed that the SLO-2 current is a major component of the delayed rectifier. We demonstrate in C. elegans that slo-2 mutants are hypersensitive to hypoxia, suggesting a conserved role for the slo-2 gene subfamily.