cDNA cloning and chromosomal localization of the human telencephalin and its distinctive interaction with lymphocyte function-associated antigen-1.

We have isolated cDNA encoding human telencephalin (TLN), a brain segment-specific neuronal adhesion molecule. Human TLN comprises an NH2-terminal signal peptide, an extracellular region with nine Ig-like domains, a single transmembrane region, and a COOH-terminal cytoplasmic tail. The NH2-terminal five Ig-like domains of TLN were closely related to those of intercellular adhesion molecules (ICAMs)-1 and −3. The TLN gene was mapped to the human chromosome 19p13.2, where the ICAM-1, −3, and −4 (LW) genes are located. Furthermore, we observed lymphocyte function-associated antigen-1 (LFA-1)-mediated adhesion of HL-60 cells on recombinant TLN protein, as well as on ICAM-1. However, the interaction of TLN with LFA-1 on HL-60 cells was divalent cation-independent and phorbol 12-myristate 13-acetate stimulation-independent. We conclude that TLN is a unique neuronal member of ICAM subgroup of the Ig superfamily and propose a novel type of interaction between the Ig superfamily molecule and integrin, which does not require the activation of integrin. TLN on the surface of telencephalic neurons may be a target molecule in the brain for LFA-1-expressing microglia and leukocytes in physiological or pathological conditions.

The most conspicuous feature of the human brain lies in its highly developed, enormously enlarged, and elaborated telencephalon (1). The telencephalon is the most rostral brain segment, which includes the cerebral neocortex, olfactory cortex, hippocampus, striatum, amygdala, septum, and olfactory bulb. These telencephalic regions take charge of higher brain functions such as memory, learning, emotion, sensory perception, and voluntary movements.
Telencephalin (TLN) 1 is a cell surface glycoprotein whose expression is confined exclusively within the telencephalon (2)(3)(4). TLN is expressed by subsets of the telencephalic neurons, but not by glial cells. In the neurons, TLN is localized to soma-dendritic membrane, but not to axonal membrane. In the course of brain development, TLN first appears around birth when the dendritic outgrowth and branching, spine formation, and synapse formation occur in the telencephalic regions. Afterwards, TLN expression persists even in adult animals. These unique expression patterns of TLN in brain segment-, neuronal subsets-, soma-dendritic membrane-, and developmental stage-specific fashions suggest that TLN may be a crucial cell surface molecule for the formation, maintenance, and plasticity of neuronal networks in the brain (2-7). We previously cloned cDNAs for rabbit and mouse TLN and demonstrated that TLN is a type I integral membrane protein belonging to the immunoglobulin (Ig) superfamily (7). Although there exist several members of the Ig superfamily that are expressed in brain region-and neuronal type-specific manners, such as limbic system-associated membrane protein (LAMP) (8), neurotrimin (9), TAG-1 (10), F3 (11), BIG-1 (12), and BIG-2 (13), TLN shows the most restricted pattern of expression. Of all the Ig superfamily molecules so far identified, the structure of TLN is most closely related to those of intercellular adhesion molecules (ICAMs)-1 and -3. The NH 2 -terminal Ig-like domain of TLN contains four cysteine residues that are capable of forming two intradomain disulfide bridges. Similarly spaced cysteine residues in the NH 2 -terminal Ig-like domains are seen in a unique subgroup of the Ig superfamily, such as ICAM-1 (14,15), ICAM-2 (16), ICAM-3 (17,18), ICAM-4 (LW antigen) (19,20), vascular cell adhesion molecule-1 (VCAM-1) (21), and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) (22). These molecules have been reported to use members of the integrin family as their counter-receptors (20,23), implicating a possible interaction of TLN with a certain integrin molecule (4).
In this paper, we cloned cDNA for human TLN, mapped its chromosomal locus, and showed an interaction of TLN with lymphocyte function-associated antigen-1 (LFA-1, CD11a/ CD18, or ␣ L ␤ 2 integrin), a common counter-receptor for ICAM-1, -2, -3, and -4 (20,23). The TLN/LFA-1 binding provides a basis for understanding molecular mechanisms underlying cell-cell interactions between telencephalic neurons and LFA-1-expressing microglia or leukocytes. * This work was supported in part by the Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan, by grants-in-aid from the Ministry of Education, Science, and Culture of Japan, and by grants from the Uehara Memorial Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To obtain full-length cDNA for human TLN, an unamplified human cerebral neocortex gt11 cDNA library (9 ϫ 10 5 recombinant phages) was screened with the 32 P-labeled PCR product (186 base pairs) as a probe. Twelve clones were randomly selected from the 50 positives of the first screening, isolated, and subcloned into the EcoRI site of pBluescript SK(ϩ) (Stratagene, La Jolla, CA). Restriction mapping and partial sequencing revealed that all 12 clones were derived from the same gene. The complete DNA sequence was determined for both strands of the longest clone hTLN-#12.
Northern Analysis-Equal amounts of total RNA from human hippocampus and cerebellum were size-fractionated in 1.5% formaldehydeagarose gel and transferred to nylon membrane (Hybond N ϩ , Amersham, UK). An ApaI fragment (828 base pairs, residues 507-1334) of human TLN cDNA was 32 P-labeled with a random priming kit (Boeh-ringer Mannheim) and used as a probe. Hybridization, washing, and visualization were performed as described previously (7).
Chromosomal Mapping by Fluorescence in Situ Hybridization-Fluorescence in situ hybridization (FISH) was carried out as described (20). In brief, human (pro)metaphase chromosomes were prepared from normal male lymphocytes using the thymidine synchronization, BrdU release technique for the delineation of G-bands. Before hybridization, chromosomes were stained in Hoechst 33258 and irradiated with UV. A 3.0-kilobase pair full-length cDNA of the human TLN was labeled with biotin-16-dUTP by nick translation and hybridized to the denatured chromosome slides at a final concentration of 25 ng/l in a mixture of 50% formamide, 10% dextran sulfate (Sigma), 2 ϫ SSC, sonicated salmon sperm DNA (2 g/l), and Escherichia coli tRNA (2 g/l). The hybridization signals were detected with FITC-avidin (Boehringer Mannheim), and chromosomes were counterstained with propidium iodide (1 g/ml). The precise signal position was determined by the delineation of G-banding patterns.
Production of Recombinant Fc-chimeric Proteins-Recombinant soluble Fc-chimeric proteins were produced essentially as described (27). Briefly, for preparation of human TLN/Fc protein, the mammalian expression plasmids were constructed with the human TLN cDNA encoding the extracellular region (a signal peptide and distal five or nine Ig-like domains) and the human IgG1 gene Fc region (28) in pEF-BOS (29). The chimeric proteins (hTLN(1-5)/Fc and hTLN(1-9)/ Fc) were produced in COS7 cells by the standard DEAE-dextran transfection method and purified from culture supernatants using a protein A-Sepharose column (Pharmacia Biotech Inc., Uppsala, Sweden). Human ICAM-1/Fc, rat BIG-1/Fc, rat BIG-2/Fc, and signal peptide/Fc (SP/Fc) were prepared by similar procedures. Cell Adhesion Assay-Cell adhesion assay was performed essentially as described (30). Immulon-3 96-well plastic plates (Dynatech, Chantilly, VA) were coated with 1 g/well goat anti-human Fc IgG (Sigma) in phosphate-buffered saline overnight at 4°C, blocked with 0.4% bovine serum albumin in phosphate-buffered saline for 2 h at room temperature, and then incubated with Fc-chimeric proteins in 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM MgCl 2 for at least 2 h at room temperature.
The HL-60 human promyelocytic leukemia cells were grown in RPMI 1640 medium supplemented with 20% fetal bovine serum. Prior to the adhesion assay, the cells were labeled for 12-24 h with [ 3 H]thymidine at 10 Ci/1 ϫ 10 6 cells, harvested, and washed three times with the assay medium (RPMI 1640 supplemented with 2.5% fetal bovine serum) prewarmed at 37°C. In order to increase avidity of LFA-1, the labeled cells were stimulated with 80 nM of phorbol 12-myristate 13-acetate (PMA, Wako, Osaka, Japan) for 30 min at 37°C. The cells were added to the Fc-chimeric protein-coated plates to give 1.5 ϫ 10 5 cells/well in a final volume of 100 l and incubated for 30 min at 37°C. Cells that remained bound after three washes were lysed in 1% SDS, and radio-activity was counted using a liquid scintillation analyzer.

RESULTS
cDNA Cloning of Human TLN-To isolate cDNA for human TLN, we performed PCR using human cerebral neocortex cDNA template and two degenerate oligonucleotide primers (Fig. 1, dashed underlines) based on the conserved amino acid sequences of mouse and rabbit TLN (7). An appropriately sized PCR product (186 base pairs) was subcloned, sequenced, and used as a probe for the following plaque hybridization. The first screening of unamplified gt11 cDNA library from human cerebral neocortex (9 ϫ 10 5 recombinants) yielded 50 positive clones. Twelve clones were randomly selected, subjected to the further screenings, and finally isolated. They showed similar restriction mapping patterns, suggesting that they are derived from the same gene. The clone #12 with the longest insert was selected, sequenced for both strands, and analyzed.
In the clone #12 (3,035 nucleotides), there are a long open reading frame of 2,772 nucleotides, a 5Ј-untranslated region of 65 nucleotides, and a 3Ј-untranslated region of 198 nucleotides, which contains a polyadenylation signal at 13 nucleotides upstream of the poly(A) tail (Fig. 1). Within the 3Ј-untranslated region there are five ATTTA motifs, which are believed to confer instability to mRNA (31,32). There is one potential translation initiation site (ATG, nucleotides 66 -68) surrounded by the nucleotides well matching with the Kozak's consensus sequence (33). An in-frame termination codon TAG locates 54 nucleotides upstream of the initiator ATG.
The human TLN polypeptide contains two hydrophobic segments ( Fig. 1, boxes), which may serve as a signal peptide (27 residues at the NH 2 terminus) and a transmembrane region (28 residues near the COOH terminus). Between the two hydrophobic regions, there is a large extracellular region (805 residues) with 15 potential N-linked glycosylation sites. A cytoplasmic region at the carboxyl terminus is 64 residues in length.
Amino acid sequence alignment of human, mouse, and rabbit TLN is shown in Fig. 2. The human TLN is 84 and 85% identical with mouse and rabbit TLN in amino acid sequence, respectively. The presence of nine Ig-like domains and the position of all cysteine residues (28 in the extracellular region and 2 in the cytoplasmic region) are completely conserved in all the three species. Twelve N-linked glycosylation sites are conserved in human, mouse, and rabbit TLN.
Northern blot analysis demonstrated human TLN mRNA of 3.0 kilobase pairs in the hippocampus; however, no signal was detected in the cerebellum (Fig. 3). This result is in good agreement with the telencephalon-specific expression of TLN (2, 3, 7).
Relationship of TLN with ICAM Subgroup Members-The extracellular region of TLN comprises nine tandemly arranged Ig-like domains, rendering TLN to a member of the Ig superfamily (34 -36). In particular, the distal eight Ig-like domains are closely related to those of ICAMs (14 -20) (Fig. 4). Total amino acid identity is 50% with ICAM-1 (domains I-V), 55% with ICAM-3 (domains I-V), 38% with ICAM-2 (domains I and II), and 32% with ICAM-4 (domains I and II). The highest homology is observed with the domain II of ICAM-1 and the domains II-IV of ICAM-3 with more than 64% amino acid identity. Together with the presence of characteristic four cysteine residues in the first domain, it will be concluded that TLN is a novel member of the ICAM subgroup of the Ig superfamily. The chromosomal localization of human TLN gene was determined using the FISH technique. The full-length cDNA of the human TLN, which did not cross-hybridize to ICAM-1 and ICAM-3 in genomic Southern blot analysis (data not shown), was used as a probe for the FISH mapping. Among 52 (pro) metaphase chromosome spreads analyzed, 16 showed twinspot signals on both homologous chromosomes 19p. Since the short arm of chromosome 19p contains mostly R-bands, it is difficult to visualize high-resolution bands by R-banding FISH. Thus, we detected FITC signals on propidium iodide-stained prometaphase chromosomes and then the sublocalization was confirmed by delineation of the high-resolution G-bands. In this system, the precise locus of the human TLN gene was localized on 19p13.2 as shown in Fig. 5. Interestingly, the genes of human ICAM-1 (37), ICAM-3 (38), and ICAM-4 (39) were also mapped to this locus, suggesting that these four molecules were derived from a common ancestral gene by gene duplication.
Adhesion of LFA-1-expressing HL-60 Cells to TLN-Of all the members of the Ig superfamily, ICAM-1, -2, -3, -4, VCAM-1, and MAdCAM-1 are characteristic in that they interact with integrin counter-receptors (20,23). The amino-terminal Ig-like domains of these six molecules have similarly arranged four cysteine residues, suggesting that formation of two intradomain disulfide bonds might be of functional importance for the interaction with integrins. These four cysteine residues are well conserved also in the first Ig-like domain of TLN, raising a possibility of the interaction of TLN with integrin(s). Furthermore, the highly conserved residues proposed to be a critical part of an integrin-binding structure in the first Ig-like domains of ICAMs and VCAM-1 (30, 40 -43) are also found in TLN (boxed region in Fig. 6). In particular, human TLN has an identical amino acid sequence in this region with human ICAM-3 and mouse ICAM-2, suggesting that TLN might interact with LFA-1 integrin, a common counter-receptor for ICAMs. To examine this possibility, we performed an adhesion assay with LFA-1-expressing HL-60 cells and purified TLN protein.
Soluble recombinant fusion proteins were produced and purified to nearly homogeneity (Fig. 7). TLN(1-5)/Fc comprises the amino-terminal five Ig-like domains of human TLN and the Fc region of human IgG1. ICAM-1/Fc, a positive control, contains all the five Ig-like domains of human ICAM-1 fused to the Fc. Signal peptide/Fc (SP/Fc), a negative control, contains the ICAM-1 signal peptide and the Fc. Cell adhesion assays were carried out with the chimeric proteins immobilized onto the plastic plates via anti-human IgG antibody.
When PMA-stimulated HL-60 cells were added into the Fcchimeric protein-coated wells and incubated at 37°C for 30 min, about 40% of the added cells bound strongly onto the TLN(1-5)/Fc-coated wells (Fig. 8B). In the same condition, about 60% of the cells bound onto the ICAM-1/Fc-coated wells (Fig. 8C). In contrast, only 5% bound nonspecifically to SP/Fc (Fig. 8A) or bovine serum albumin (data not shown), suggesting that the Fc portion is not responsible for the adhesion. The HL-60 adhesion was dependent on the amount of coated proteins (Fig. 9). A similar result of the HL-60 adhesion was obtained when TLN(1-9)/Fc, a fusion protein consisting of the whole extracellular region of TLN and the IgG1 Fc region, was used as a substrate (data not shown). Fc-chimeric proteins of the other two neuronal Ig superfamily molecules, BIG-1 (12) and BIG-2 (13), did not support the adhesion of HL-60 cells (data not shown), indicating that the binding of HL-60 cells to TLN is specific.
It has been reported that most of the interaction mediated by integrins are dependent on the presence of divalent cations in the extracellular milieu and that the activation of integrinexpressing cells induce conformational change of the integrins from a low-to high-avidity form (44 -46). These two properties hold true for the binding between ICAM-1 and LFA-1. As shown in Fig. 11, the addition of EDTA completely abolished the HL-60 adhesion to ICAM-1/Fc, and the pretreatment of HL-60 cells with PMA resulted in marked enhancement of binding to ICAM-1/Fc. On the other hand, the HL-60 cells behaved in a different manner with regard to the adhesion to TLN/Fc. First, EDTA hardly influenced the HL-60 adhesion to TLN/Fc, indicating no requirement of divalent cations for the binding between TLN and LFA-1. Second, the pretreatment with PMA did not increase the HL-60 adhesion to TLN/Fc, FIG. 4. Structural comparison of TLN with ICAM-1, -2, -3, and LW antigen (ICAM-4). A, amino acid sequence alignment of TLN with ICAMs and LW antigen. The amino acid sequence of human TLN (domains I-V) was aligned with those of ICAM-1 (domains I-V) (8,9), ICAM-2 (domains I and II) (10), ICAM-3 (domains I-V) (11,12), and LW antigen (domains I and II) (13). The open circles denote the highly conserved Cys residues. The residues conserved in more than three of the five members (domains I and II) or more than two of the three (domains III-V) are highlighted. B, proposed model of the domain structure of TLN, ICAMs, and LW antigen. Amino acid identities between the corresponding Ig-like domains of TLN, ICAMs, and LW antigen are shown with Arabic numerals above the Ig-like domains, respectively. Individual Ig-like domains are numbered I-IX on the top. Extra-and intracellular orientations are indicated by NH 2 and COOH on the protein chains, respectively. Intradomain disulfide bonds are represented by SS.
indicating that the binding between LFA-1 and TLN is activation-independent. These results suggest that LFA-1 on HL-60 cells is constitutively activated for TLN binding, but not for ICAM-1, or that TLN can bind to both resting (low-avidity) and active (high-avidity) forms of LFA-1 on HL-60 cells. DISCUSSION We have identified and characterized the human TLN. TLN is a type I integral membrane protein with nine Ig-like domains in its extracellular region, belonging to the Ig superfamily. Here, we provided three characteristic features of TLN that indicate an intimate relationship of TLN with ICAMs. First, among all the Ig superfamily molecules so far identified, the Ig-like domains of TLN are most closely related to those of ICAM-1 and ICAM-3 and also show a weaker but significant homology to those of ICAM-2 and ICAM-4. Second, chromosomal locus of the human TLN gene was mapped to 19p13.2 in the vicinity of other three ICAM genes (ICAM-1, -3, and -4). Third, TLN protein was able to bind LFA-1, a common counterreceptor for ICAMs, although the manner of interaction was distinctive (see below). These findings indicate that TLN is a member of ICAM subgroup of the Ig superfamily and shares many properties with hitherto known ICAM subgroup members. However, the expression of TLN is confined to the telencephalon of the central nervous system (2, 3, 7), whereas all the other ICAM members are expressed mostly by cells in the immune and blood systems, such as lymphocytes (ICAM-1 and -3), endothelial cells (ICAM-1 and -2), epithelial cells (ICAM-1), and erythrocytes (ICAM-4) (14 -19). Thus, although the molec-  The human TLN is highly homologous to mouse and rabbit TLN with 85% amino acid identity. This value is significantly higher than those between human and mouse ICAM-1 (53%) (47) or ICAM-2 (60%) (48), reflecting difference in molecular evolution between the nervous and immune systems. Compared with neurons in the brain which are sequestered within the blood-brain barrier, cells in the immune system suffer from more direct attacks by pathogens such as viruses and parasites that invade into blood and lymph streams. It has been assumed that pathogens frequently target immunologically important structures such as cell surface molecules. In particular, several adhesion molecules in the immune system have been identified as viral or parasitic receptors: ICAM-1 for rhinovirus and malaria-infected erythrocytes (49 -51), CD4 for human immunodeficiency virus (52), Mac-1 (␣ M ␤ 2 integrin) for hookworm glycoprotein (53), and ␣ V integrins for adenovirus (54). Therefore, the cell adhesion molecules in the immune system may undergo more frequent changes in their molecular structures in order to escape from viral attacks, resulting in lower amino acid identities among different species. This is in good agree-ment with the idea by Murphy (55) that the proteins in host defense system have changed their sequences more rapidly in evolution in response to the challenge by pathogens.
We observed significant and specific adhesion of HL-60 cells on purified recombinant TLN/Fc, as well as on ICAM-1/Fc. The adhesion on TLN/Fc was inhibited by pretreatment of HL-60 cells with two mAbs against CD11a (G43-25B, 38) and one mAb against CD18 (YFC118.3). This result indicates that LFA-1 is a candidate counter-receptor for TLN.
In contrast, three mAbs (HI-111, MHM-24, MHM-23) that completely blocked the LFA-1/ICAM-1 binding had no effect on the binding of HL-60 cells to TLN/Fc. Conversely, a mAb G43-25B strongly inhibited the LFA-1/TLN binding, whereas it showed little effect on the LFA-1/ICAM-1 binding. The overlapping but different profile of these mAbs in adhesion blocking activity indicates that the binding site on LFA-1 for TLN may be close to but distinct from that for ICAM-1. Similar differential effects of a panel of anti-LFA-1 mAbs were reported in T cell binding to ICAM-1 versus ICAM-3 (56). Thus, LFA-1 is capable of binding its multiple counter-receptors in a selective manner.
The binding of LFA-1 on the HL-60 cells to TLN/Fc was unusual in that it required neither divalent cations nor PMA stimulation. Most of the interactions mediated by integrins are dependent on divalent cations and are activated by inside-out signaling (44,45) with a few exceptions (57,58). In the case of LFA-1 integrin, four different counter-receptors act in distinctive manners in respect of requirement for divalent cations, activation by phorbol ester, and activation by anti-CD44 antibody (Table I) (59). Our result suggests that LFA-1 on the HL-60 cells may be constitutively activated for the binding to TLN, but not to ICAM-1. A similar activation-independent  binding has been reported in the case of interaction between ICAM-3 and a novel leukointegrin, ␣ d ␤ 2 (58).
In the central nervous system, LFA-1 is constitutively expressed by microglia, but not by neurons, astrocytes, nor oligodendrocytes (60,61). The microglia are also called brain-type macrophages, usually ramified in a resting state, but activated in neurological diseases including Alzheimer's disease, acquired immune deficiency syndrome, and brain trauma. When activated, the microglia change their shape, migrate to the damaged neurons, proliferate, show neurotoxic activity, and remove dead cells by phagocytosis (62,63). The constitutive expression of TLN in neurons and LFA-1 in microglia and the present finding demonstrating TLN/LFA-1 binding without integrin activation suggest that TLN and LFA-1 may mediate neuron/microglia interaction in the telencephalon of the normal brain. This interaction might be necessary for the central nervous system to hold microglia on a tight leash, suppressing its phagocytic and cytotoxic activity in the healthy brain. Alternatively, TLN might be a neuronal target molecule for LFA-1-expressing activated microglia or infiltrating neutrophils, T lymphocytes, and macrophages in some brain diseases. Thus, the finding of binding between TLN and LFA-1 provides a basis for detailed understanding of the molecular mechanisms underlying cell-cell interactions between telencephalic neurons and LFA-1-expressing leukocytes and microglia. Such knowledge could help to investigate possible molecular tools that prevent LFA-1-expressing leukocytes and activated microglia from attacking and destroying telencephalic neurons in neurological diseases.