Ectodomain shedding of L1 adhesion molecule promotes cell migration by autocrine binding to integrins.

The L1 adhesion molecule plays an important role in axon guidance and cell migration in the nervous system. L1 is also expressed by many human carcinomas. In addition to cell surface expression, the L1 ectodomain can be released by a metalloproteinase, but the biological function of this process is unknown. Here we demonstrate that membrane-proximal cleavage of L1 can be detected in tumors and in the developing mouse brain. The shedding of L1 involved a disintegrin and metalloproteinase (ADAM)10, as transfection with dominant-negative ADAM10 completely abolishes L1 release. L1-transfected CHO cells (L1-CHO) showed enhanced haptotactic migration on fibronectin and laminin, which was blocked by antibodies to alpha v beta 5 and L1. Migration of L1-CHO cells, but not the basal migration of CHO cells, was blocked by a metalloproteinase inhibitor, indicating a role for L1 shedding in the migration process. CHO and metalloproteinase-inhibited L1-CHO cells were stimulated to migrate by soluble L1-Fc protein. The induction of migration was blocked by alpha v beta 5-specific antibodies and required Arg-Gly-Asp sites in L1. A 150-kD L1 fragment released by plasmin could also stimulate CHO cell migration. We propose that ectodomain-released L1 promotes migration by autocrine/paracrine stimulation via alpha v beta 5. This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions.     

Structural insights into chemokine CCL17 recognition by antibody M116

The homeostatic chemokine CCL17, also known as thymus and activation regulated chemokine (TARC), has been associated with various diseases such as asthma, idiopathic pulmonary fibrosis, atopic dermatitis and ulcerative colitis. Neutralization of CCL17 by antibody treatment ameliorates the impact of disease by blocking influx of T cells. Monoclonal antibody M116 derived from a combinatorial library shows potency in neutralizing CCL17-induced signaling. To gain insight into the structural determinants of antigen recognition, the crystal structure of M116 Fab was determined in complex with CCL17 and in the unbound form. Comparison of the structures revealed an unusual induced-fit mechanism of antigen recognition that involves cis-trans isomerization in two CDRs. The structure of the CCL17-M116 complex revealed the antibody binding epitope, which does not overlap with the putative receptor epitope, suggesting that the current model of chemokine-receptor interactions, as observed in the CXCR4-vMIP-II system, may not be universal.     

Trimerization of cell adhesion molecule L1 mimics clustered L1 expression on the cell surface: influence on L1-ligand interactions and on promotion of neurite outgrowth

Several studies indicate that cell adhesion molecules have to be clustered on the cell surface to engage in adhesive functions. We investigated adhesive functions of clustered versus monomeric L1 extracellular parts in vitro to distinguish how clustering affects ligand binding and promotion of neurite outgrowth. Trimeric L1 was recombinantly expressed and covalently assembled by the cartilage matrix protein's coiled-coil domain. Trimeric L1 has an apparent molecular mass of approximately 380 kDa in the nonreduced form and approximately 130 kDa in the reduced form. Rotary shadowing electron micrographs of trimeric L1 revealed a rod-like shape terminating in three globular domains. Monomeric L1 assumes a horseshoe shape of domains Ig I-IV followed by a rod-like structure consisting of Ig V and VI and fibronectin type III 1-5. Circular dichroism measurements showed that the secondary structure consists of beta-sheets. Trimeric L1 binds to itself, to monomeric L1, to laminin-1, and to alpha5beta1 integrin in a concentration-dependent manner. In contrast, binding of monomeric L1 could only be saturated with itself but not with laminin-1 and with alpha5beta1 integrin. Promotion of neurite outgrowth from PC12 cells cultured on adsorbed trimeric L1 was increased by 100%, whereas on monomeric L1 the increase was only 50% over the control value. Promotion of neurite outgrowth from PC12 cells was specifically inhibited in a concentration-dependent manner by a polyclonal antibody against L1. These findings show that clustering of only three extracellular domains increases considerably L1's binding affinity to different ligands and enhances neurite outgrowth, suggesting that adhesive functions of L1 on the cell surface depend on cluster formation.     

Elevated phenylalanine levels interfere with neurite outgrowth stimulated by the neuronal cell adhesion molecule L1 in vitro

Elevated levels of phenylalanine (Phe) as observed in patients with phenylketonuria interfere with proper neuronal development, leading to severe psychomotor deficits and mental retardation. We have analyzed the effects of Phe on neurite outgrowth in vitro. When expressed in fibroblasts, the neuronal cell adhesion molecules L1 and plexin B3 strongly increase the length of neurites emanating from cerebellar neurons in co-culture experiments. Elevated Phe blocks L1-mediated, but not plexin B3-mediated outgrowth, whereas tyrosine is ineffective. Elevated Phe also interferes with aggregation of fibroblasts overexpressing L1, suggesting that the pathological effect of elevated Phe occurs by interfering with L1-mediated cell adhesion.     

Ethanol inhibits L1 cell adhesion molecule activation of mitogen-activated protein kinases

Inhibition of the functions of L1 cell adhesion molecule (L1) by ethanol has been implicated in the pathogenesis of the neurodevelopmental aspects of the fetal alcohol syndrome (FAS). Ethanol at pharmacological concentrations has been shown to inhibit L1-mediated neurite outgrowth of rat post-natal day 6 cerebellar granule cells (CGN). Extracellular signal-related kinases (ERK) 1/2 activation occurs following L1 clustering. Reduction in phosphoERK1/2 by inhibition of mitogen-activated protein kinase kinase (MEK) reduces neurite outgrowth of cerebellar neurons. Here, we examine the effects of ethanol on L1 activation of ERK1/2, and whether this activation occurs via activation of fibroblast growth factor receptor 1 (FGFR1). Ethanol at 25 mm markedly inhibited ERK1/2 activation by both clustering L1 with cross-linked monoclonal antibodies, or by L1-Fc chimeric proteins. Clustering L1 with subsequent ERK1/2 activation did not result in tyrosine phosphorylation of the FGFR1. In addition, inhibition of FGFR1 tyrosine kinase blocked basic fibroblast growth factor (bFGF) activation of ERK1/2, but did not affect activation of ERK1/2 by clustered L1. We conclude that ethanol disrupts the signaling pathway between L1 clustering and ERK1/2 activation, and that this occurs independently of the FGFR1 pathway in cerebellar granule cells.     

Ethanol causes the redistribution of L1 cell adhesion molecule in lipid rafts

Fetal alcohol spectrum disorder is estimated to affect 1% of live births. The similarities between children with fetal alcohol syndrome and those with mutations in the gene encoding L1 cell adhesion molecule (L1) implicates L1 as a target of ethanol developmental neurotoxicity. Ethanol specifically inhibits the neurite outgrowth promoting function of L1 at pharmacologic concentrations. Emerging evidence shows that localized disruption of the lipid rafts reduces L1-mediated neurite outgrowth. We hypothesize that ethanol impairment of the association of L1 with lipid rafts is a mechanism underlying ethanol's inhibition of L1-mediated neurite outgrowth. In this study, we examine the effects of ethanol on the association of L1 and lipid rafts. We show that, in vitro, L1 but not N-cadherin shifts into lipid rafts following treatment with 25 mM ethanol. The ethanol concentrations causing this effect are similar to those inhibiting L1-mediated neurite outgrowth. Increasing chain length of the alcohol demonstrates the same cutoff as that previously shown for inhibition of L1-L1 binding. In addition, in cerebellar granule neurons in which lipid rafts are disrupted with methyl-beta-cyclodextrin, the rate of L1-mediated neurite outgrowth on L1-Fc is reduced to background rate and that this background rate is not ethanol sensitive. These data indicate that ethanol may inhibit L1-mediated neurite outgrowth by retarding L1 trafficking through a lipid raft compartment.     

Ontogeny of the cell adhesion molecule L1 in the cerebellum of weaver and reeler mutant mice

The ontogeny of cell adhesion molecule L1 in cerebellum was quantitatively assessed in weaver and reeler mutant mice and in heterozygous litter-mate controls. In the latter the concentration and the amount of L1 both increased from the first postnatal week to become maximum at the second. In contrast, in the weaver and reeler neurologic mutant mice, L1 decreased steadily. The L1 concentration and the amount of L1 was lower in the cerebellum of homozygous mutant mice than in litter-mate controls. The findings are consistent with L1 being a component of axonal plasma membranes. However, no evidence was found of any direct effect of thewv andrl phenotypes on L1 expression.     

Characterization of recombinant soluble carcinoembryonic antigen cell adhesion molecule 1

Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a type 1 transmembrane, homotypic cell adhesion protein expressed on epithelial and hematopoietic cells. CEACAM1 has four major isoforms with three or four immunoglobulin (Ig)-like ectodomains and either long or short cytoplasmic domains. In a 3D model of breast epithelial cell morphogenesis, CEACAM1 plays an essential role in lumen formation [J. Cell Sci. 112 (1999) 4193]. Two soluble ectodomain isoforms of CEACAM1 expressed in myeloma cells were immunologically active and highly glycosylated. The molecular weights of the 3-ecto- and 4-ectodomain isoforms were 90 and 110kDa, respectively, and monomers by sedimentation equilibrium centrifugation. Both isoforms were prolate ellipsoids with axial ratios of 6 for the 3-ecto- and 8 for 4-ectodomain isoforms, respectively, by size exclusion chromatography and analytical ultracentrifugation. Both isoforms caused a significant reduction in lumen formation when tested in the 3D model culture system.     

Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.     

AlphaII-spectrin participates in the surface expression of cell adhesion molecule L1 and neurite outgrowth

AlphaII-spectrin, a basic component of the spectrin-based scaffold which organizes and stabilizes membrane microdomains in most animal cells, has been recently implicated in cell adherence and actin dynamics. Here we investigated the contribution of αΙΙ-spectrin to neuritogenesis, a highly complex cellular process which requires continuous actin cytoskeleton remodeling and cross-talk between extracellular cues and their cell surface receptors, including cell adhesion molecules. Using RNA interference-mediated gene silencing to down-regulate αΙΙ-spectrin expression in human neuroblastoma SH-SY5Y cells, we observed major changes in neurite morphology and cell shape: (1) reduced mean length and a higher number of neurites per cell; occasional long neurites were thinner and displayed abnormal adhesiveness during cell migration resulting in frequent breaks; similar persisting adhesiveness and breaks were also observed in trailing edges of cell bodies; (2) irregular polygonal cell shape in parallel with loss of cortical F-actin from neuronal cell bodies; (3) reduction in protein levels of αΙ- and βΙ-spectrins, but not βΙΙ-spectrin (4) decreased global expression of adhesion molecule L1 and spectrin-binding adapter ankyrin-B, which links L1 to the plasma membrane. Remarkably, αΙΙ-spectrin depletion affected L1 – but not NCAM – cell surface expression, and L1 clustering at growth cones. This study demonstrates that αΙΙ-spectrin is implicated in normal morphology and adhesive properties of neuron cell bodies and neurites, and in cell surface expression and organization of adhesion molecule L1.     

Structural characterization of blood group A glycosphingolipids recognized by the antibody 3G9-A

In this study, the antibody 3G9-A was assayed for activity against human erythrocyte glycosphingolipids. The antibody was found to recognize glycosphingolipid components from blood group A erythrocytes but not glycosphingolipids from blood group B or O erythrocytes. Subsequent investigation revealed that the glycosphingolipid components recognized by the antibody were also recognized by a blood group A specific monoclonal antibody. The structures of two of the isolated active glycosphingolipid components were structurally characterized using proteon nuclear magnetic resonance (¹H NMR) and gas chromatography-mass spectrometry (GC-MS) techniques and were found to consist of two blood group A glycosphingolipids; the type 2 chain A^b and type 3 chain A^a glycosphingolipids. Subsequent analysis of the remaining active components by GC-MS and immunostaining techniques revealed that all of the active components were blood group A glycosphingolipids. Furthermore, structural studies of the active components suggested that the epitope of the antibody consisted of the group A trisaccharide, GalNAc1,3(Fuc1,2)Gal.Abbreviations GC-MS gas chromatography-mass spectrometry - ¹H NMR proton nuclear magnetic resonance - Gal d-galactose - Glc d-glucose - Fuc l-fucose - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - Cer ceramide - mAb monoclonal antibody - BSA bovine serum albumin - PBS phosphate buffered saline - FID free induction decay - PMAA partially methylated alditol acetates     

Generation of affinity matured scFv antibodies against mouse neural cell adhesion molecule L1 by phage display

The recognition molecule L1 plays important functional roles in the nervous system and in non-neural tissues. Since antibodies to L1 are of prime importance to study its functional properties, we have generated affinity matured human single chain variable fragment (scFv) antibodies against mouse L1 by introducing random mutations in the complementarity determining regions (CDRs) of a previously isolated scFv antibody heavy chain (CDR1 and CDR2) and light chain (CDR3). After biopanning the mutant library, a clone (5F7) that gave the strongest ELISA signal was expressed, purified, and characterized. The dissociation constant of 5F7 (2.86 x 10(-8)M) was decreased 60-fold compared to the wild type clone G6 (1.72 x 10(-6)M). 5F7 detected L1 by Western blot analysis in mouse brain homogenates and recognized L1 in L1 transfected cells and cryosections from mouse retina and optic nerve by immunofluorescence. Bivalent 5F7 scFv antibody (5F7-Cys) was also generated and showed a dissociation constant of 5.22 x 10(-9)M that is 5.5-fold lower than that of monomeric 5F7 antibody. The bivalent affinity matured L1 scFv antibody thus showed stronger binding by a factor of 310 compared to the wild type clone. This antibody should be useful in various biological assays.     

Two alcohol binding residues interact across a domain interface of the L1 neural cell adhesion molecule and regulate cell adhesion

Ethanol may cause fetal alcohol spectrum disorders (FASD) in part by inhibiting cell adhesion mediated by the L1 neural cell adhesion molecule. Azialcohols photolabel Glu-33 and Tyr-418, two residues that are predicted by homology modeling to lie within 2.8 Å of each other at the interface between the Ig1 and Ig4 domains of L1 (Arevalo, E., Shanmugasundararaj, S., Wilkemeyer, M. F., Dou, X., Chen, S., Charness, M. E., and Miller, K. W. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 371–375). Using transient transfection of NIH/3T3 cells with wild type (WT-L1) and mutated L1, we found that cysteine substitution of both residues (E33C/Y418C-L1) significantly increased L1 adhesion above levels observed for WT-L1 or the single cysteine substitutions E33C-L1 or Y418C-L1. The reducing agent β-mercaptoethanol (βME) reversibly decreased the adhesion of E33C/Y418C-L1, but had no effect on WT-L1, E33C-L1, or Y418C-L1. Thus, disulfide bond formation occurs between Cys-33 and Cys-418, confirming both the close proximity of these residues and the importance of Ig1-Ig4 interactions in L1 adhesion. Maximal ethanol inhibition of cell adhesion was significantly lower in cells expressing E33C/Y418C-L1 than in those expressing WT-L1, E33C-L1, or Y418C-L1. Moreover, the effects of βME and ethanol on E33C/Y418C-L1 adhesion were non-additive. The cutoff for alcohol inhibition of WT-L1 adhesion was between 1-butanol and 1-pentanol. Increasing the size of the alcohol binding pocket by mutating Glu-33 to Ala-33, increased the alcohol cutoff from 1-butanol to 1-decanol. These findings support the hypothesis that alcohol binding within a pocket bordered by Glu-33 and Tyr-418 inhibits L1 adhesion by disrupting the Ig1-Ig4 interaction.     

Immunocytological characterization of the expression of cell adhesion molecule L1 during early innervation of mouse otocysts

Summary Doubts exist as to whether afferent nerve fibers exert a neurotrophic effect on the differentiation of sensory cells in the developing vestibular neuroepithelium. To determine whether innervation of hair cells precedes their differentiation, we have used the L1 adhesion molecule as a marker for axons. The detection of L1 on afferent axons in the otic vesicle of mouse embryos on gestation day 11 shows that nerve fibers penetrate the neuroepithelium before the sensory cells differentiate. L1-immunoreactivity of nerve endings also reveals the considerable fiber ramification on gestation days 14 and 15, i.e., corresponding to the first stages of sensory cell differentiation. The expression of L1 at successive stages of nerve fiber growth in the neuroepithelium, such as fasciculation and ramification, is not consistent with the previous role proposed for L1 as a fascicule-promoting factor and raises the possibility that other mechanisms are involved in L1 mediaded adhesion.     

Properties of a goat anti-L antibody Further evidence for heterogeneity of the L antigen

The preparation and properties of an antibody (anti-L) against low potassium type (LK) goat red cells raised in a high potassium type (HK) goat are described. This reagent stimulated active potassium transport, but showed only weak serological activity against low potassium type (LK) sheep and goat red cells. The results are discussed in relation to the hypothesis that anti-L anti-body has two specificities — a sodium pump-stimulating activity (anti-Lp) and a serological activity (anti-L_ly.     

Isolation and characterization of human monoclonal antibodies specific to MMP-1A, MMP-2 and MMP-3

Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are up-regulated in many diseases, but the use of MMP inhibitors for therapeutic purposes has often been disappointing, possibly for the limited specificity of the drugs used in clinical trials. In principle, individual MMPs could be specifically drugged by monoclonal antibodies, either by inhibition of their catalytic activity or by antibody-based pharmacodelivery strategies. In this article we describe the isolation and affinity maturation of recombinant antibodies (SP1, SP2, SP3) specific to the murine catalytic domains of MMP-1A, MMP-2 and MMP-3. These novel reagents allowed a systematic comparative immunofluorescence analysis of the expression patterns of their cognate antigens in a variety of healthy, cancerous and arthritic murine tissues. While all three MMPs were strongly expressed in tumor and arthritis specimens, MMP-1A was completely undetectable in the normal tissues tested, while MMP-2 and MMP-3 exhibited a weak expression in certain normal tissues (e.g., liver). The new antibodies may serve as building blocks for the development of antibody-based therapy strategies in mouse models of pathology.     

Crystal structure of human sulfotransferase SULT1A3 in complex with dopamine and 3'-phosphoadenosine 5'-phosphate

The human sulfotransferase, SULT1A3, catalyzes specifically the sulfonation of monoamines such as dopamine, epinephrine, and norepinephrine. SULT1A3 also has a unique 3,4-dihydroxyphenylalanine (Dopa)/tyrosine-sulfating activity that is preferentially toward their D-form enantiomers and can be stimulated dramatically by Mn2+. To further our understanding of the molecular basis for the unique substrate specificity of this enzyme, we solved the crystal structure of human SULT1A3, complexed with dopamine and 3'-phosphoadenosine 5'-phosphate, at 2.6 A resolution and carried out autodocking analysis with D-Dopa. The structure of SULT1A3 enzyme-ligand complex clearly showed that residue Glu146 can form electrostatic interaction with dopamine and may play a pivotal role in the stereoselectivity and sulfating activity. On the other hand, residue Asp86 appeared to be critical to the Mn2+-stimulation of the Dopa/tyrosine-sulfating activity of SULT1A3, in addition to a supporting role in the stereoselectivity and sulfating activity.     

Acetic acid activates PKD1L3-PKD2L1 channel—A candidate sour taste receptor

The polycystic kidney disease (PKD) 1L3-PKD2L1 channel is a candidate sour taste receptor expressed in mammalian taste receptor cells. Various acids are reported to activate PKD channels after the removal of the acid stimuli, but little information is available on the activation of these channels by acetic acid. It was difficult to analyze the PKD channel activation by acetic acid using Ca²⁺ imaging experiments because this acid induces a transient and nonspecific response in cultured cells. Here, we developed a novel method to evaluate PKD channel activation by acetic acid. Nonspecific responses were observed only over a short period after the application of acetic acid. In contrast, PKD channel activation evoked by acetic acid as well as citric acid was detected even at a later time point. This method revealed that PKD1L3-PKD2L1 channel activation by acetic acid was pH-dependent and occurred when the ambient pH was <3.1.     

Rsu1 contributes to cell adhesion and spreading in MCF10A cells via effects on P38 map kinase signaling

The ILK, PINCH, Parvin (IPP) complex regulates adhesion and migration via binding of ILK to β1 integrin and α?parvin thus linking focal adhesions to actin cytoskeleton. ILK also binds the adaptor protein PINCH which connects signaling proteins including Rsu1 to the complex. A recent study of Rsu1 and PINCH1 in non-transformed MCF10A human mammary epithelial cells revealed that the siRNA-mediated depletion of either Rsu1 or PINCH1 decreased the number of focal adhesions (FAs) and altered the distribution and localization of FA proteins. This correlated with reduced adhesion, failure to spread or migrate in response to EGF and a loss of actin stress fibers and caveolae. The depletion of Rsu1 caused significant reduction in PINCH1 implying that Rsu1 may function in part by regulating levels of PINCH1. However, Rsu1, but not PINCH1, was required for EGF-induced activation of p38 Map kinase and ATF2 phosphorylation, suggesting a Rsu1 function independent from the IPP complex. Reconstitution of Rsu1-depleted cells with a Rsu1 mutant (N92D) that does not bind to PINCH1 failed to restore FAs or migration but did promote IPP-independent spreading and constitutive as well as EGF-induced p38 activation. In this commentary we discuss p38 activity in adhesion and how Rsu1 expression may be linked to Map kinase kinase (MKK) activation and detachment-induced stress kinase signaling.