Lazarillo, a new GPI-linked surface lipocalin, is restricted to a subset

INTRO

Embryopathwathe appmillionnervougrowthtion of and phy(BixbyTessier-Lavigne, 1992; Goodman and Shatz, 1993). Becauseneurons in the same embryonic environment often followdifferent pathways, we believe that the interaction of a growthcone with its environment requires a code of molecular signalsand receptors to provide the necessary specificity. The ‘labeledpathways hypothesis’ (Goodman et al., 1982) proposes amechanism for this specificity: axon fascicles in the embryonicnervous system are differentially labeled by surface moleculesthat are used for the guidance of growth cones. Many surfacemolecules restricted to subsets of axon fascicles have beenfound in both vertebrates and invertebrates. They belong toseveral families of proteins such as the immunoglobulin super-family, the cadherins, some members of the integrin family,and others (reviewed by Bixby and Harris, 1991; Goodman andShatz, 1993).

The lipocalins are a family of extracellular soluble proteinsthat transport small hydrophobic molecules. They have a wellconserved structure: a calyx formed by a

β-barrel with ahydrophobic ligand pocket. This family includes proteins with

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lipocalin family and has a renervous system that makes it a candidate for a specific axonalreceptor and/or guidance cue. In the following paper (Sánchez,Ganfornina and Bastiani, 1995) we indeed demonstrate that itis required for the navigation of identified commissuralneurons. To the best of our knowledge, the localization,molecular characteristics and function of this new lipocalin areunique, both among the members of this protein family andamong the proteins involved in axonal pathfinding during thedevelopment of the nervous system. For its putative role inaxon guidance, we have named this protein Lazarillo after themain character of a sixteenth century Spanish novel,

Lazarillode Tormes, a crafty boy who guided a blind man.

MATERIAL AND METHODS

Grasshopper (Schistocerca americana) embryos were obtained froma colony maintained at 31°C and 60% humidity at the University of

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Biology

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For example, the serum retinol-ers of vitamin A from the liver toff et al., 1990); C8γ is one of theent cascade (Haefliger et al.,

aglandin D synthetase (PGDs) istivity (Nagata et al., 1991); theposed to regulate cell differen-a-Renevey et al., 1989). Within

there has been no indication thatrocess of axonal pathfinding.

cribe in this work belongs to thestricted expression pattern in the

123

defines Lazarillo as a membertracellular carriers of smallost related to the porphyrin-s. Lazarillo is the first example

the plasma membrane, highlyd to a subset of developing

, GPI, grasshopper, lipid-binding

o a subset of

nt 121, 123-134 (1995)reat Britain © The Company of Biologists Limited 1995

ARY

rillo, a new GPI-linked surface lipocalin, is restricted

ons in the grasshopper embryo

. Ganfornina*,†, Diego Sánchez* and Michael J. Bastiani

Department, University of Utah, Salt Lake City, UT 84112, USA

M. D. G. contributed equally to this workr correspondence

DUCTION

nic neurons must extend growth cones along preciseys, often over long distances, to reach and synapse withropriate target cell. The proper execution of this task bys of neurons is essential to construct a functionals system. What cues are used by a neuron to direct its cone toward a distant target? Since the first observa-growth cones by Ramón y Cajal, a variety of chemicalsical cues have been invoked as guidance mechanisms

and Harris, 1991; Cypher and Letourneau, 1992;

diverse biological functiobinding proteins are transpvarious target tissues (Blocomponents of the com1987); the human brain pra lipocalin with enzymatimouse oncogene 24p3 istiation and mitogenesis (Hthis large and diverse famany are involved in thHowever, the protein we

llo, a protein recognized by the monoclonal antibodys expressed by a subset of neurons in the developings system of the grasshopper. It is a glycoprotein ofMr with internal disulfide bonds and linked to thellular side of the plasma membrane by a glycosyl-atidylinositol moiety. Peptide sequences obtainedffinity purified adult protein were used to identifybryonic cDNA clone, and in situ hybridizationsed that the distribution of the Lazarillo mRNA par-

that of the monoclonal antibody labeling on

embryos. Sequence analyof the lipocalin familyhydrophobic ligands, anand retinol-binding lipocof a lipocalin anchored glycosylated, and restrneurons.

Key words: pathfinding, lipoproteins

124

Utah. Theaccording10E6 waembryoni(Hockfielddescribed

ImmunoEmbryoniby Seaverand electrwashed wgelatin, 0.dry milk antibody incubated2 hours, µCi/ml inwas air dr

BiochemTo analyzand/or bmembranepH 11.3, 1mM NaClThe mixttrifuged (1ciated pro

Analysidescribed RPMI meculture mMedium (Gibco-BRincubated,U/ml in mphosphatidsis, generophosphatidBoehringewashed, fiof proteinsbation waEmbryos protein pr

To detpurified pcellulose temperatuNaCl, 0.1for 1 houTTBS-MnVectastaindeglycosyFlavobactprotein wand then iControl prenzyme. Iwith 0.15Sigma).

Protein pproteinThe protesoluble fra

M

y were staged by percentage of embryonic development to Bentley et al. (1979). The monoclonal antibody (mAb)s generated by Carpenter and Bastiani (1990) againstc nervous tissue using a subtractive immunization method, 1987). Immunocytochemistry was carried out as

by Sánchez, Ganfornina and Bastiani. (1995).

blot analysis of embryonic and adult proteinsc and adult membrane proteins were prepared as described et al. (1991), separated by electrophoresis (SDS-PAGE)otransferred to nitrocellulose membranes. Membranes wereith a Tris buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 0.2%1% NaN3) and blocked for 2 hours with 2.5% BSA, 2.5%in the previous buffer. They were incubated with primary(1:200 in blocking solution) for 2 hours, washed again, with rabbit anti-mouse IgG (1:500 in blocking solution) forwashed and incubated with 125I-protein A solution (0.2 blocking solution) for 1 hour. After washing, the membraneied and exposed to film.

described (Bastiani et al., 1987), using mAb 10E6 immobilized toProtein G Sepharose beads (Pharmacia). Lysate total protein concen-tration was measured with the micro-BCA assay (Pierce). Adult lysatewas prepared as described for embryos but using whole adultgrasshoppers whose gut, legs and wings were removed. Amino acidcomposition of Lazarillo was performed by transferring 2.7 µg ofprotein to PVDF membrane (Problott) followed by acid hydrolysisand amino acid analysis.

To obtain peptide sequence 13.5 µg of the protein Lazarillo wereseparated by SDS-PAGE. The gel piece containing the protein wasrinsed in 250 mM Tris, pH 9.0, 250 mM EDTA and in dH2O at roomtemperature, chopped and dried in a vacuum centrifuge. The dried gelwas soaked in 0.1 M NH4HCO3, pH 9.0 containing endoproteinaseLys-C (Lys-C from Lysobacter enzymogenes, Boehringer-Mannheim), and incubated at 37°C for 12 hours (enzyme to proteinratio, 1:20). Digestion products were eluted from the gel by extensivewashing with NH4HCO3 buffer. Peptides were then separated byreverse phase HPLC using a C-18 300 Å pore column. Fractions weresubjected to microsequencing by automated Edman degradation.

. D. Ganfornina, D. Sánchez and M. J. Bastiani

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Amino acid analysis, peptide separation and sequencing, as well as
ical characterization of Lazarillo protein the association of Lazarillo with the membrane, high saltsic pH extractions were carried out. Unsolubilized proteins were subjected to a basic pH buffer (10 mM TEA,50 mM NaCl), a high salt buffer (10 mM TEA, pH 7.8, 500 or a combination of both conditions for 30 minutes on ice.re was diluted 10 times with the same buffer and cen-00,000 g, 2 hours). Both supernatant and membrane asso-eins were analyzed by immunoblot.
s of GPI-anchoring to the membrane was performed asy Chang et al. (1992). 45% embryos were dissected in sterileium containing 6 mg/ml glycine. They were transferred to

edium (Cellgro) containing 50% Schneider’s Drosophilaibco-BRL), 49% Minimum Essential Medium (α medium,

L), 1% antibiotic-antimycotic solution (Sigma). They were without (control embryos) or with phospholipase C (PLC) 3dium, for 2 hours at 31°C. Two different enzymes were used,ylinositol-specific PLC (PI-PLC, from Bacillus thuringien-usly provided by Dr Martin Low, Columbia University) orylcholine-specific PLC (PC-PLC, from Bacillus cereus,r-Mannheim). After the incubation period embryos wereed and labeled with antibodies. For the immunoblot analysis

extracted by the treatment, the culture medium after the incu- collected and concentrated with Centricon-10 (Amicon).ere washed, then homogenized and a standard membrane

paration was followed as above.

synthesis of custom oligonucleotide primers for PCR or DNAsequencing were carried out at the Protein/DNA Core Facility of theUtah Cancer Center under the direction of R.W. Schackmann.

Molecular analysis of Lazarillo cDNADegenerate oligonucleotides were designed from the peptidesequences to amplify DNA fragments from embryonic grasshoppercDNA using PCR with Taq DNA polymerase (Saiki et al., 1988). PCRwas conducted in a thermal cycler (Perkin Elmer Cetus) and con-ditions were as follows: MgCl2 concentration 2.5 mM; one cycle of94°C for 2 minutes; 35 cycles of 94°C for 30 seconds, 48°C for 30seconds, and 72°C for 1 minute; and a final cycle of 72°C for 5minutes. The DNA fragments obtained were tested with a pair ofinternal primers that were designed to give a known size DNAfragment. A PCR product of approx. 400 bp, DL400, was cloned intopCR-II vector using the TA cloning system (Invitrogen) andsequenced (see below). The fragment DL400 was radiolabeled with[32P]dCTP using the random primer method (Prime-It II kit, Strata-gene) and used as a probe.

Several embryonic cDNA grasshopper libraries were constructedand screened with the DL400 probe as described in the λZAP system(Stratagene). We screened 0.5×106 plaque forming units (p.f.u.) froman amplified library made using nerve cords dissected from 55%embryos, and 1.3×106 p.f.u. from an unamplified library made fromembryos at 45% development.

Both strands of cDNA inserts were sequenced (Sanger et al., 1977)
rmine whether Lazarillo is a glycoprotein, the affinityotein was separated by SDS-PAGE and transferred to nitro-embrane. Blots were preincubated for 30 minutes at room
e in TTBS-Mn-Ca buffer (0.1 M Tris, pH 7.5, 150 mM Tween-20, 0.1 mM MnCl2, 0.1 mM CaCl2) and incubated

r with biotinilated concanavalin A (Vector) 10 µg/ml in-Ca solution. All subsequent steps were as described in ABC kit (Vector). In addition, affinity purified protein waslated with peptide-N-glycosidase F (PNGase F) fromerium meningosepticum (Boehringer-Mannheim). Thes denatured with 10 mM β-mercaptoethanol and boilingcubated with 3.3 U/ml of PNGase F at 37°C for 18 hours.

otein was denatured in the same way, but incubated withoutn parallel, pure protein was heat denatured and incubatedmU/ml of neuraminidase (from Arthrobacter ureafaciens,

urification and microsequencing of Lazarillo

n was purified by affinity chromatography from detergentctions of embryonic or adult lysates, prepared as previously

using Sequenase (v2.0, U.S. Biochemicals) and custom primers. dITPwas employed to sequence through areas with GC compressions.DNA and protein sequences were analyzed with the GCG programs(Devereux et al., 1984) and the BLAST service (Altschul et al., 1990).The following data bases were screened: Swissprotein, PIR,GenBank/EMBL, and Brookhaven PDB. Sequence alignements werecarried out with PILEUP GCG program using a symbol comparisontable that takes into account structurally conservative amino acid sub-stitutions (Risler et al., 1988).

Analysis of Lazarillo mRNA expressionTotal RNA was prepared from grasshopper embryos at 45% devel-opment. 500 embryos were dissected in cold RPMI with 6 mg/ml ofglycine, washed in PBS, and homogenized in 0.1 M Tris, pH 7.5, 4M guanidinium thiocyanate, 1% β-mercaptoethanol. The RNA waspurified by CsCl centrifugation as described in Sambrook et al.(1989). Poly(A)+ mRNA was isolated from total RNA using the Poly-ATract mRNA isolation system (Promega) and analyzed by northernblot analysis with the DL400 probe. Briefly, 5 and 10 µg mRNA wereseparated by electrophoresis in a formaldehyde-agarose gel, trans-ferred to a nylon membrane (Zeta-Probe, BioRad) under alkaline con-

ditions,as for li

In sitgrasshocation).4 kit, Bclone ahydroly(37% foMgSO4gradualSSC, 2550 µg/min DEPC55°C inwas the36-48 hhybridizphatasereactiondetectio

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The mrestricThe exproteinsubset cones, binds tof labpathwaThey dembryocontactthe fasganglioexpressdevelopscaffolpc) con(lc) linThe grathe persegmenshows eral senexpressmotonearrow).

The comparrestrictFig. 1DFasciclto the fphorin GoodmnectivefascicleLazaril

125Lazarillo: a neuronal GPI-linked lipocalin

and hybridized with the probe at 65°C in the same solutionbrary screenings.u hybridization was performed according to a protocol forpper whole-mount embryos (J. Broadus, personal communi- Briefly, a digoxigenin-11-dUTP labeled RNA probe (Genius-oehringer-Mannheim) was synthesized using the entire Laz-5s template. The resulting RNA was subjected to alkalinesis. Embryos were dissected and fixed in PEM-formaldehydermaldehyde 1:9 in 0.1 M Pipes, pH 6.9, 2 mM EGTA, 1 mM) for 50 minutes. After washing, the solution was changedly to hybridization solution (50% deionized formamide, 4×0 µg/ml yeast tRNA, 500 µg/ml boiled salmon sperm DNA,l heparin, 0.1% Tween- 20, 1× Denhardts, 5% dextran sulfate-treated H2O) at room temperature, with a last incubation at

hybridization solution for 1 hour. The labeled RNA proben added at 0.5 µg/ml and incubation proceeded at 55°C forours. Washes for 5 hours, with frequent changes, followed theation step. To detect the labeled RNA, an alkaline phos-

-conjugated anti-digoxigenin antibody was used and the color was carried out as described in the Genius DNA labelling and

the vMP2 fascicle and some axons in this fascicle expressLazarillo through adulthood. A coronal section of an adult lon-gitudinal connective labeled by mAb 10E6 is shown in theinset of Fig. 1D. Lazarillo shares with Semaphorin I itsexpression on the MFT, however, semaphorin I is restricted tothe initial portion of the bifurcated axons while Lazarillo islocated along the entire axonal length (Fig. 1E), reflecting adifferent spatial distribution. Two bundles in the posteriorcommissure are labeled by Fasciclin II and Lazarillo, butFasciclin II expression is transient while that of Lazarilloremains, reflecting a different temporal regulation. Theexpression of the four molecules in distinct but overlappingsubsets of pathways supports the hypothesis that a particularcombination of molecules on the surface of a fascicle functionsas the unique label used by neurons for their specific pathwaydecisions.

Lazarillo is also expressed by a subset of neuroblasts in theCNS, every sensory neuron in the PNS, a group of neurons of

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b 10E6 recognizes a surface moleculeted to a subset of neurons and axon fasciclesression of the antigen recognized by mAb 10E6, theLazarillo, is restricted to the surface of an identifiedf neurons. It is detected on cell bodies, axons, growthnd filopodia of live embryos suggesting that the mAb an extracellular surface epitope. Fig. 1A shows a pairled neurons that pioneer an anterior commissural at 32% of embryonic development (the AcP cells).

irect their growth cones toward the midline of the while extending many filopodia ahead (arrow) tothe contralateral growth cone. Fig. 1B shows some oficles labeled with the mAb 10E6 in a metathoracic

n at 40% of development. Unlabeled axon fascicles, noting Lazarillo, are indicated by open arrows. At thismental stage pioneer neurons have established a of axon fascicles consisting of two commissures (ac,necting the hemiganglia, two longitudinal connectivesing adjacent ganglia, and a median fiber tract (MFT).

the enteric nervous system (ENS) and by a few non-neuronaltissues. The developmental expression pattern is described inthe following paper (Sánchez, Ganfornina and Bastiani, 1994).

Lazarillo is a glycoprotein anchored to themembrane by a glycosyl-phosphatidylinositol groupWe identified Lazarillo by immunoblot analysis of embryonicmembrane proteins separated by SDS-PAGE in the presenceof reducing agents. A smeary band with an average apparentMr of 45×103 is recognized by the mAb 10E6 (Fig. 2A, lane2). In the absence of the mAb the secondary antibody recog-nizes a nonspecific band of 57×103 Mr (Fig. 2A, lane 3) thatalso appears when other unrelated mAbs are used as theprimary antibody. No signal was detectable in the solublefraction of embryonic homogenates (not shown). A band ofsimilar appearance and Mr was also detected by immunoblotanalysis using adult membrane preparations (Fig. 2A, lane 4).The apparent Mr of Lazarillo does not change significantlyunder non-reducing conditions (Fig. 2B). Lazarillo waspurified from embryonic lysates on a mAb 10E6 affinitycolumn (Fig. 2A, lane 5) with a yield of approx. 1 ng/mg oftotal protein.

Several lines of evidence suggest that Lazarillo is associatedwith membranes, in addition to the 10E6 labeling obtained in

sshopper central nervous system (CNS) is connected topheral nervous system (PNS) by two main nerves; thetal (SN) and intersegmental (ISN) nerves. Fig. 1Che ISN, where only the motoneurons (mn) and periph-sory neurons (sn) navigating along its posterior branch Lazarillo. The anterior branch, pioneered by the Uurons, are never labeled by the mAb 10E6 (open

fascicles expressing Lazarillo were identified anded with those expressing other surface moleculesd to subsets of fascicles in the grasshopper embryo. In the distribution of Lazarillo is compared with that ofn I. Fig. 1E represents the 10E6 fascicle map comparedscicles expressing Fasciclin I, Fasciclin II, and Sema-

I/Fasciclin IV (Bastiani et al., 1987; Harrelson andan, 1988; Kolodkin et al., 1992). The longitudinal con- at 40% of development are composed of three distincts differentially labeled by these surface glycoproteins.o is currently the only one on the surface of axons in

non-permeabilized live embryos. The mAb recognizes theprotein in the membrane but not in the soluble fraction ofembryonic homogenates. Neither high salt, alkaline solutions,nor both combined, extract Lazarillo from the membranefraction (not shown) suggesting that the protein is associatedwith the membrane by hydrophobic interactions. To determinewhether Lazarillo is linked to the membrane by a GPI moiety,embryos were treated with PI-PLC prior to fixation andlabeling with the mAb 10E6. Fig. 3 shows the results fromcontrol (A,B) and treated embryos (C,D). Following PI-PLCtreatment, no signal was obtained with the mAb 10E6 in theCNS or in the PNS. As a control, we labeled embryos withmAb 3B11, which recognizes the GPI-anchored glycoproteinFasciclin I (Hortsch and Goodman, 1990). As expected, thelabeling disappeared after the enzymatic treatment. In contrast,when treated embryos were labeled with 8C6, a mAb againstFasciclin II, which has a transmembrane domain in grasshop-pers (Harrelson and Goodman, 1988), the immunoreactivitydid not change after PI-PLC treatment (not shown). To test the

126 M. D. Ganfornina, D. Sánchez and M. J. Bastiani

Fig. 1. Lazarillo expression is restricted to a specific subset of neurons and axon fascicles in the embryonic nervous system of the grasshopper.(A-D) Presence of Lazarillo in the CNS visualized by immunocytochemistry with mAb 10E6. Whole embryos are viewed from the dorsalsurface with DIC optics. Anterior is up. (A) The identified pioneer neurons of the anterior commissure (AcP cells) are labeled with mAb 10E6in a subesophageal segment at 32% of embryonic development. Each neuron sends a growth cone (arrowhead) toward the midline where it willencounter its contralateral homologue. Filopodial extensions are darkly stained with this antibody. The arrow points to a long filopodiumalmost reaching the contralateral growth cone. (B) Thoracic ganglion at 40% of development. Only a subset of axon bundles is labeled.Compare the labeled medial longitudinal fascicle (solid arrow) with the unlabeled lateral longitudinal fascicles (open arrow). lc, longitudinalconnectives; ac, anterior commissure; pc, posterior commissure; SN, segmental nerve. (C) The intersegmental nerve (ISN) exits betweenadjacent segmental ganglia. The growth cone of a sensory neuron (sn) finds the axon of a motoneuron (mn), both labeled by mAb 10E6, andenters the CNS by the posterior branch of the ISN. The anterior branch, or U fascicle, is not labeled by mAb 10E6 (open arrow). The midline isindicated by a dashed line. (D) Double labeling with mAb 3B11 (anti-Fasciclin I, brown) and mAb 10E6 (blue). Three distinct longitudinalfascicles are present at 38% of development that are differentially labeled with these surface molecules. Lazarillo is present only in the vMP2fascicle (arrows) while Fasciclin I is in a lateral longitudinal fascicle (including the A/P fascicle) and the U fascicle (arrowheads). Theunlabeled one (open arrow) is the MP1/dMP2 fascicle. Midline is on the right. The inset shows a transverse section of an adult longitudinalconnective showing a ventral median tract labeled by mAb 10E6. Ventral is down and midline on the left. (E) Schematic diagram showing thecomplete subset of fascicles that expresses Lazarillo at 40% (on the left) compared to Fasciclin I, Fasciclin II and Semaphorin I expression (onthe right). MFT, median fiber tract; pISN, posterior branch of ISN; aISN, anterior branch of the ISN (or U fascicle); SN, segmental nerve; A/P,MP1/dMP2, vMP2, Q, L and RP1 are fascicles named after the neurons that pioneer them. Scale bars: A-C and inset in D, 50 µm; D, 25 µm.

127Lazarillo:

specificity of the effects of PI-PLC, we treated embryos withPC-PLC. None of the labeling (10E6, 3B11 or 8C6) decreasedafter the treatment (not shown) suggesting that the conditionsused selectively remove GPI-anchored proteins and do notcause nonspecific release or damage of membrane proteins.Accordingly, the 45×103 Mr band is present only in themembrane preparation from control embryos, but is entirelyreleased to the culture supernatant after treatment with PI-PLC(Fig. 3E). The nonspecific band (Mr, 57×103) is not extractedby PI-PLC. These results suggest that the 45×103 Mr protein islinked to the membrane by a GPI tail and corresponds to theLazarillo pattern observed in embryos.

The smeary appeacould be the result opresence of carbohycovalently linked tpurified Lazarillo icanavalin A. The lecshown) suggesting tresidues. As the lattlinked oligosacchariwhich cleaves N-lin40×103 Mr is seen 4A,B). Our interpre

Fig. 5×103.(A) I . (1)Coom ide gelshow ns. (2)The roteinmigr ow) inthe e In theabse yreco r. (4) Asingl e thanthe e izedwith fromadul rifiedby a iclysat rotein

does not affect its electrophoretic mobility. Lane R: the electrophoresis sample buffer contains β-m educingagent added. Molecular mass markers (Mr×10−3) are shown on the left.

Fig. 3. A glycosyl-phosphatidylinositol group anchors Lazarillo to the extracellular side of the plaand leg from a control embryo at 45% of development, labeled with mAb 10E6. (C,D) The labelinthe presence of a phosphatidylinositol-specific phospholipase C (PI-PLC) for 90 minutes. (E) Memthe incubation media (Sup) were analyzed by immunoblot in control and PI-PLC-treated embryoscompletely released from the membranes by the enzyme. The nonspecific band (open arrow) remaµm; (B,D) 200 µm. Molecular mass markers in Mr×10−3 are shown on the left in E.

a neuronal GPI-linked lipocalin

2. Lazarillo is a protein with a Mr of 4mmunoblot analysis using mAb 10E6assie blue-stained SDS-polyacrylam

ing total embryonic membrane proteimAb 10E6 specifically recognizes a pating at an apparent Mr of 45×103 (arrmbryonic membrane preparation. (3) nce of the mAb the secondary antibodgnizes a nonspecific band of 57×103 Me band of the same size and appearancmbryonic 45×103 Mr protein is recogn the mAb on total membrane proteins t grasshopper. (5) Lazarillo protein puffinity chromatography from embryones. (B) The redox state of the native percaptoethanol (50 mg/ml), NR: no r

-PAGEe to theharidesubating Con-in (notannose of N-ase F,

of 30-r (Fig.due to

rance of the band seen under SDSf incomplete SDS solubilization dudrates. The presence of oligosacco Lazarillo was assayed by incmmobilized on nitrocellulose withtin does bind to the 45×103 Mr protehe presence of D-glucose and D-mer are present in the core structuredes, we treated the protein with PNGked carbohydrates. A diffuse bandabove a tight band of 28.2×103 Mtation is that this result could be

sma membrane. (A,B) Metathoracic gangliong disappears when embryos are cultured inbrane proteins (MP) and soluble proteins in

. The 45×103 Mr protein (solid arrow) isins in the membrane fraction. Bars, (A,C) 50

128

partial dethe 28.2×protein. AmAb 10ELazarillo.linked olitide. Thehydrolyzecharides, Lazarillo

Given trophoretshown inbilizationcatenary PNGase Fdeglycosytrophoretsistent wicore prot

Generatto a singLazarillo the immochoice fong/mg osequenceanalysis peptides peptides (3) RPDxSVHFPNundetermthreoninein peptidasparagin

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Fig. 4. A large shift in relative mobility is obLazarillo after deglycosylation and subsequenof reducing agents. (A) Silver stained gel sho45×103 Mr protein purified from adult grasshoaffinity chromatography (Control). DeglycosPNGase F (Deglyc.) generates a smeary bandfrom 30-40×103 Mr with a sharp lower bound28.2×103 Mr. The enzyme band (Mr, 35.5×10in the third lane for comparison. (B) Analysisimmunoblot of the same samples. The mAb 1recognizes the protein after removal of the Ncarbohydrates (Deglyc.). In this case the loweof the smear is resolved into a tight band of 2that we think represents the fully deglycosylaThe upper band is due to antibody leaking froaffinity column. (C) Lazarillo protein wasdeglycosylated in the absence of reducing ageelectrophoretic mobility tested under non-red

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and reducing (R) conditions. Only a small proportion ofced core

glycosylation of the native protein and suggests that103 Mr band represents the fully deglycosylateds revealed by immunoblot analysis (Fig. 4B) the6 is able to recognize the deglycosylated form of This suggests that the 10E6 epitope is not on the N-gosaccharides, but could be part of the core polypep-protein was also treated with neuraminidase, whichs the terminal bonds joining sialic acid to oligosac-and no change in electrophoretic mobility of

was observed (not shown).the large influence of the carbohydrates on the elec-c mobility of Lazarillo, the effect of reducing agentsFig. 2B is probably masked by the anomalous solu- of the glycoprotein. To analyze the presence of intra-disulfide bonds we deglycosylated Lazarillo with in the absence of β-mercaptoethanol (Fig. 4C). Thelated protein shows a clear decrease in elec-c mobility when the reducing agent is present, con-th the presence of internal disulfide bonds in Lazarilloin.

portions of peptide 3. A DNA fragment of appro(DL400) was amplified by PCR from embryonic gcDNA using the sense primer from peptide 2 and thprimer from the C-terminal end of peptide 3. An opframe (ORF) was found along the entire DNA fragmadditionally contained peptide 4. The sequence conthe mRNA coding for the peptides obtained fgrasshoppers is present in embryos. The undetermacids referred to as possible N-glycosylation sites wasparagines.

The DL400 DNA was used as a probe for northization (Fig. 5B). A single band of approximatedetected in a poly(A)+ mRNA preparation from e45% of development, a stage that corresponds wexpression as seen by immunocytochemistry with t

Isolation of Lazarillo cDNA clone. The prediamino acid sequence has the characteristicGPI-linked membrane protein We screened several λZAP embryonic grasshop

is deglycosilated due to the low efficiency of the PNGase F in these conditions (native protein not shown). The non-redugrates as a 23×103 Mr band. Molecular mass markers (Mr×10−3) are shown on the left.

ion of a Lazarillo DNA probe. Hybridizationle species of embryonic mRNAprotein was purified from adult lysates by affinity tobilized mAb 10E6. Adult lysates were the source ofr the protein purification due to their larger yield (15f total protein). Attempts to obtain N-terminal

were unsuccessful. An amino acid compositionwas performed and we chose Lys-C to produceof reasonable length. After HPLC separation, fourwere sequenced: (1) NLQLDLNK; (2) WYEYAK;SAASTEISWILLRSRxSSxMTLERVEDELK; (4)SPSVGNYxILSTDYDxYSIV; where x stands for

ined amino acids. The presence of a serine or a two residues after some undetermined amino acidses 3 and 4 suggested that the latter would bees bound to oligosaccharides (N-linked).airs of degenerate primers (sense and antisense) werefrom peptide 2, and from the N- and C-terminal

libraries with the DL400 probe and identified three positiveclones. Two of them (Laz-9 and Laz-10) were identical clonesfrom an amplified nerve cord specific library. They contain aninsert of 2.8 kb and were truncated at the 5′ end. The third clone(Laz-5) was from an unamplified library and contains an insertof 3075 bp that corresponds in size to the mRNA detected bynorthern hybridization. Both DNA strands of the Laz-5 insertwere sequenced. Sequences corresponding to the four peptidesare contained in this clone. The cDNA and the predicted aminoacid sequence of Laz-5 are shown in Fig. 5A. It contains a 5′untranslated region (UTR) of 137 bp, an ORF of 642 bp, anda 3′ UTR of 2296 bp. The sequence of the four peptides areunderlined. The DL400 probe hybridizes to bp 273-653.

The first methionine codon encountered in frame in the Laz-5 clone is in an appropriate context to be the translationinitiation site (Cavener and Ray, 1991). The length of the ORFagrees with the analysis of period three compositional con-straint (TESTCODE GCG program) and the codon usage andcompositional bias in the third position of each possible codon


(CODOtable mfound

Boththis Orepresetransloreticulment tis indibeforeHeijne

Fig. 5.(A) Nuthe righlinked Some oPoly(Atransferthe predthe sign

or a GPI-linked membrane glycoprotein and is represented by a single embryonic transcript.ce of Laz-5 cDNA clone. Nucleotide numbers are on the left and amino acid numbers are onheads point to the potential cleavage sites of the signal peptide and GPI tail. The potential N-

s. Cysteine residues are circled. The peptides obtained by Lys-C digestion are underlined.

The sequence of Lazarillo cDNA codes fcleotide and predicted amino acid sequent (referred to the first methionine). Arrow

glycosylation sites are noted by black dot

NPREFERENCE GCG program with a codon usageade from Schistocerca and Locusta coding sequences

in the cited data bases). the N- and C-terminal ends of the protein predicted by

RF are hydrophobic (Fig. 5C). These fragments maynt, respectively, the signal peptide necessary for thecation of the protein into the lumen of endoplasmicum and the hydrophobic tail necessary for the attach-o a GPI group. The predicted cleavage site for Lazarillocated in Fig. 5A before Ala22, though it could also be Gln23. In both cases the rule ‘-3,-1’ described by von (1990) is fulfilled. The 16 C-terminal amino acids of

Laz-5 are hydrophobic, and preceded by a hydrophilic spacerdomain (Glu195 to Val198). Both domains define the potentialGPI-anchoring signal with a cleavage site before or afterAla192 (Coyne et al., 1993). The predicted mature proteinafter cleavage of both signal sequences would have a Mr ofapproximately 20×103. The deduced protein has sevenpotential N-glycosylation sites (Fig. 5A and C), some of themindeed glycosylated as deduced from the experiments usingPNGase F (Fig. 4). The four cysteine residues present, notedin Fig. 5A, can form internal disulfide bonds that wouldexplain the different electrophoretic mobility produced byreducing agents (Fig. 4C). Four potential polyadenylation

f the polyadenylation sites and mRNA instability sequences are shown double underlined. (B) Northern hybridization analysis.)+ mRNA (5 and 10 µg) from embryos at 45% of development was hybridized with DL400 probe after electrophoretic separation and to a nylon membrane. A single transcript of approximately 3 kb is detected. Size markers in kb are on the left. (C) Hydropathy plot oficted protein sequence determined by the Kyte and Doolittle method (1982) using a window of 9 residues. The hydrophobic domains ofal peptide and the GPI-anchoring signal are noted by the filled pattern. Black dots represent glycosylation sites.

130

signals athe 3′ UT

LazarillantigenThe spatiforming embryosusing thhybridizaof the samis detecte6A). Thprimary ing with 6B). The specificalthe LazaSeveral cmetathoraing sensotribution other areatures charand subesin embryocating thadue to spThese exp10E6, themRNA re

LazarilloThe dedu

M

ig. 6. mRNA and proteino-localization inrasshopper embryosonfirms the identity of theaz-5 clone. (A,B) Anteriorortion of whole-mountmbryos at 32% showinghe head and subesophagealegments. The mRNA (A)nd the protein (B)olocalize, for example, inhe AcP cells in the S2egment (arrowheads). Inhe head the four labeled

the metath(C) and th


FcgcLpetsactst

entified it as a memberins; extracellular carriers

nce similarity to mostth its closest relatives aalues commonly foundl., 1993), and a 72-85%tructurally conservativeaterial and Methods).tertiary structure despite

ily. Flower et al. (1993)nce relationships withinsing the known three-s they have defined three3) that correspond to the

lls in A correspond to thenes extending axons alonge commissure and thengitudinal connectives ofe brain in B. (C,D) Thensory groups of cells ineen the Lazarillo mRNA

nd four message instability sequences are found inR (Fig. 5A).

o mRNA colocalizes with the mAb 10E6

al localization of Lazarillo mRNA was studied by per-in situ hybridization in whole-mount grasshopper

. A digoxigenin-labeled RNA probe was generatede entire Laz-5 cDNA. A comparison of the RNAtion pattern with the mAb 10E6 labeling in embryose age is shown in Fig. 6. Lazarillo mRNA expression

d in a group of neuronal cell bodies in the head (Fig.ese same neurons, whose axons contribute to thecommissure of the brain and to the fascicles connect-the segmental ganglia, are labeled with the mAb (Fig.

several data bases and the searches idof the family of proteins called lipocalof small hydrophobic ligands.

Lazarillo shows significant sequemembers of this family. It shares wiresidue identity between 19-30%, vbetween family members (Flower et asimilarity when taking into account samino acid substitutions (see MLipocalins share a highly conserved the low identity values found in the famhave analyzed the structure and sequethe lipocalin family. By superimpodimensional structure of four lipocalinstructurally conserved regions (SCR1-

ceothlothse

oracic legs of embryos at 40% of development (arrowheads) are shown as examples of the correlation betwe protein (D) in the PNS. Bar, 200 µm.

AcP cells of the second subesophageal (S2) segment,ly labeled by the mAb, also show hybridization withrillo RNA probe (arrowhead in Fig. 6A and B).lusters of cells expressing Lazarillo are seen in thecic leg (Fig. 6C), which corresponds to the develop-ry organs recognized by the mAb (Fig. 6D). The dis-of Lazarillo mRNA matches that of the protein in alls of the CNS and PNS, and in other embryonic struc-acteristically labeled by mAb 10E6, such as the ENSophageal body (not shown). Hybridization is absents exposed to the sense RNA probe (not shown), indi-t the signal we observe with the anti-sense probe isecific hybridization with the endogenous mRNA.eriments suggest that the antigen recognized by mAb surface glycoprotein Lazarillo, is encoded by thepresented in the cDNA clone Laz-5.

belongs to the lipocalin familyced Lazarillo protein sequence was compared with

sequence motifs highlighted in Fig. 7A. They have proposedthese SCRs as an essential requirement for belonging to thelipocalin family. Fig. 7A shows a multiple alignment ofLazarillo with the family members bearing the highest per-centage identities. The three SCRs are largely conserved inLazarillo, and other sequence stretches also show a consider-able similarity.

The size and diversity of the lipocalin family is growing sig-nificantly; currently over 60 different proteins have been iden-tified as family members. They can be further divided intoseveral non-orthologous groups according to structural featuresthat might reflect common functional properties, such as ligandbinding specificities or interactions with other proteins (Cowanet al., 1990; Peitsch and Boguski, 1990). Because of this het-erogeneous family organization we assessed whether Lazarillobelongs to a particular clade of lipocalins in order to form ahypothesis about its functional properties. A multiple sequencealignment with all lipocalins present in the data bases wasperformed. The dendrogram representing the clustering order


employmost rerelatedwas usthese ptree waclade cproteinthe por

Fig. 7. Lazarillo belongs tothe lipocalin family.(A) Multiple alignment ofLazarillo mature protein withseveral members of thelipocalin family. Thealignment was done with agap weight of 3 and a lengthweight of 0.5 (see Materialand Methods). Black boxeshighlight residue identitywith Lazarillo with athreshold of 40%. Grayboxes highlight positionswith structurally conserved

amino acid substitutions witha threshold of 60%. Thestructurally conservedregions defining the lipocalinfamily are indicated (SCR1,2 and 3). Cysteine residuesconserved among Lazarilloand its closest relatives areindicated by black triangles.The residues indicated byasterisks are in closeproximity to thehydrophobic ligand in Bbp,Icya, and Apd.(B) Phylogram of selectedmembers of the lipocalinfamily derived from protein

sequence alignments conducted as in A. A heuristicsearch of the most parsimonious unrooted tree wascarried out with the taxa shown. The total tree length is1396, with a consistency index of 0.81 and a retentionindex of 0.58. Calibration bar equals a branch length of50 residue changes. Bbp: bilin-binding protein fromPieris brassicae; Icya: Insecticyanin A from Manducasexta; Apd: human and rat Apolipoprotein D; Cra2: A2subunit of Crustacyanin from Homarus gambarus;Retb: serum retinol-binding protein from humans and

ed in the alignment was used to choose the proteinslated to Lazarillo and also examples of more distantlylipocalins. The program PAUP 3.0s (Swofford, 1991)ed to find an unrooted phylogenetic reconstruction ofroteins by a heuristic search approach. A single optimals found (Fig. 7B) that clearly relates Lazarillo with theomposed of Apolipoprotein D (Apd), Bilin-binding

(Bbp) and Insecticyanin A (Icya). This clade is calledphyrin-binding lipocalins because Bbp and Icya bind

biliverdin IXγ (Holden et al., 1987; Huber et al., 1987) and Apdcan bind bilirubin (Peitsch and Boguski, 1990). The internalphylogenetic organization of the two main branching lineageswas further analyzed using an exhaustive search procedure,including in each case a member of the opposite group. Thisstudy confirmed the basic branching pattern within each groupand that Lazarillo belongs to the Apd+Bbp+Icya clade. Toevaluate the reliability of the inferred tree, a Bootstrap analysiswas also conducted that revealed that Lazarillo is linked to a

Xenopus laevis; Ret1: serum retinol-binding proteinfrom Oncorhynchus mykiss; Purp: chicken Purpurin;24p3: mouse oncogene product; PGDs: human brainGSH independent prostaglandin D synthetase; Co8g: γsubunit of C8 component of complement cascade; Hc:human α-2-microglobulin; Mup5: mouse major urinaryprotein 5; Lacb: bovine β-lactoglobulin.

132

node coproteins

Lipocbarrel sformingbetweencontribustructure1987) hathe BbpThis infoistics of sequencsmall gabetweensecondamethod the relatshown). pattern oidenticalshare amwhich abridges affect thcharactethree proand poteLazarilloknown asterisksseven otthe commare possfeatures porphyri

DISCUS

The biocas a highbonds amembraof a growand partvertebraEspecialChang elinked pbud of thpioneer proteinstaking ppotentiathrough the memsoluble has beenhave evidetectab

M

mmon to Apd+Bbp+Icya plus the retinol-binding (Rbp) in 87% of 103 replicates (not shown).alins share a highly conserved folding pattern: a β-tructure composed of eight antiparallel β-strands two orthogonal sheets with a hydrophobic pocket in. Two α-helices at the N- and C-terminal regions alsote to the main scaffold of the protein. The tertiarys of Bbp (Huber et al., 1987) and Icya (Holden et al.,ve been resolved and that of Apd was modeled from

and Icya coordinates (Peitsch and Boguski, 1990).rmation makes it possible to predict some character-

Lazarillo secondary structure based on homology. Thee alignment of Lazarillo, Apd, Bbp and Icya containsps or insertions in Lazarillo that fall within loops

β-strands or α-helices in the other lipocalins. Thery structure of Lazarillo estimated with the statisticalof Chou and Fasman (1974) closely aligns with that of

GPI linkage to the membrane was performed at 35-45% ofdevelopment, we cannot rule out that a temporal regulation ofrelease exists later in development or in adulthood.

Lazarillo protein sequence shows significant similarity tomembers of the lipocalin family, lipid-binding proteins thatshare a tertiary structure consisting of a β-barrel scaffold witha hydrophobic ligand binding pocket. The lipocalins aredivided into several clades reflecting their diversity offunctions and ligand specificities. We performed a phyloge-netic analysis to assess which particular clade of proteinsincludes Lazarillo and found that it is associated with theporphyrin-binding lipocalins. Further comparisons revealedthe conservation of important features: the main scaffold of β-strands and α-helices is consistently predicted by homology,the pattern of cysteines forming disulfide bonds is conserved,as well as some of the amino acids participating in salt bridgesand involved in ligand interaction. All these data unequivocally


ed lipocalins whose tertiary structure is known (not

fl

recer

n

to h

ib

n

h

nn

itelt

roes alal Gbc dle

establish Lazarillo as a new member of the lipocalin family.
The four cysteines participating in the conserved alternating disulfide bonds in Bbp, Icya and Apd arey aligned in Lazarillo (Fig. 7A). Bbp, Icya and Apdino acids that participate in four salt bridges, two of also present in Lazarillo. Disulfide bonds and saltontribute to the position of certain β-strands that size of the hydrophobic pocket. Furthermore, the
istics of the binding pocket are conserved among theteins, including the location of hydrophobic patchestial hydrogen bonds (Peitsch and Boguski, 1990).shares the six most conserved of the 18 residues be in close proximity to the ligand (noted within Fig. 7A), and has conservative substitutions iner amino acids. We propose that Lazarillo containson structural core of the lipocalins, the SCRs, whichly involved in protein-protein interactions, and thosecritical for the ligand binding specificity of the-binding clade.

SION

emical properties we have analyzed define Lazarilloly glycosylated small protein, with internal disulfide

However, Lazarillo has several properties that are uniqueamong lipocalins. Lazarillo is the most glycosylated of itsclade and possibly of the lipocalin family. Only a fewlipocalins are reported to be glycosylated, and none as exten-sively as Lazarillo (Urade et al., 1989; McConathy andAlaupovic, 1976). The presence of abundant glycosylationmight be modulating the interactions of Lazarillo with othermolecules in ways different from the rest of the lipocalins. TheGPI tail links Lazarillo to the extracellular side of the plasmamembrane and prevents it from being an extracellular carrierof hydrophobic ligands as is commonly the case for lipocalins.PGDs is the only other example of a lipocalin associated withmembranes, the endoplasmic reticulum and nuclearmembranes, but it is readily dissociated from them in theabsence of detergents (Urade et al., 1985). Finally, therestricted tissue distribution of Lazarillo does not have aparallel in the lipocalin family. There are few lipocalins asso-ciated with the nervous system, and none of them is confinedto a subset of developing neurons. PGDs is first expressed indeveloping neurons and then changes to oligodendrocytes inthe adult animal (Urade et al., 1987). Apd is expressed in avariety of tissues, but accumulates at injured peripheral nerveswhere it is proposed to help remove toxic heme metabolitesproduced by hemorrhage (Boyles et al., 1989). Finally,

d linked to the extracellular surface of the plasmae by a GPI tail. The GPI linkage is a common featureing number of proteins expressed during developmentcipating in a variety of biological processes in boths and invertebrates (Cross, 1990; Ferguson, 1992).

y significant in this context are the experiments by al. (1992) that demonstrate that removal of GPI-teins from the surface of cells in the developing limb grasshopper causes several pathfinding errors by the

ensory axons. Thus, alone or in concert, GPI-linkedre certainly contributing to the signaling processesce between growth cones and their environment. Away to regulate cell-cell or cell-matrix interactionsPI-linked proteins is to be able to release them fromranes at appropriate times and locations. Although a

leaved form of the GPI-linked molecule Fasciclin Iproposed (Hortsch and Goodman, 1990), we do notence for a released form of Lazarillo, at least at levels by immunoblot analysis. Since our analysis of the

Purpurin is a Rbp secreted by retinal photoreceptor cells thatis involved in cell adhesion and transport of retinoids (Schubertet al., 1986; Berman et al., 1987).

Lipocalins are the extracellular elements of a traffickingsystem for small hydrophobic molecules that also includes thefatty acid-binding proteins (FABP), mainly located in thecytoplasm (Matarese et al., 1990), and nuclear receptors, suchas those for retinoic acid that are involved in transcriptionalregulation (Petkovich et al., 1987). Although different biolog-ical roles are attributed to these proteins, such as lipid transportand metabolism, evidence is accumulating that they functionin signaling systems, regulating various developmentalprocesses (Ross, 1993). The recently described brain lipid-binding protein (BLBP) is a neural-specific member of theFABPs whose expression is correlated with neuronal differen-tiation and is proposed to be required for the establishment ofthe radial glial fibers and the migration of cerebellar granulecells (Feng et al., 1994). In addition, cellular retinol andretinoic acid-binding proteins, CRBPs and CRABPs respec-

tively, neuronFABPsplay a al., 199pods arcamouflal., 198

The functioGanforLazariltory ofthe indescribses aboused fohydropLazarilSCRs osurfacelipocallateral of Lazahas be1992; adhesiofunctiothis codimerset al., homopby bindLazarilligand would for all of polybeen reet al., 1

To dnervouproteinhydropout linklipocalcandidabound of smato the guidanways ocone ntional n

It is oBiologyscript; Madvice; Lark fosequenc


could be involved in the guidance of spinal commissurals (Maden and Holder, 1991). Some members of the have been reported in invertebrates and proposed torole in nervous system morphogenesis (Muehleisen et3). However, the lipocalins described so far in arthro-e mainly circulating transporters of pigments related toage, photoprotection and photoreception (Holden et7; Clarke et al., 1990; Huber et al., 1987).restricted expression, membrane localization, and thenal results described in the following paper (Sánchez,nina and Bastiani, 1995) suggest a role for the lipocalinlo in the signaling events necessary to direct the trajec- growing axons in the grasshopper embryo. Based uponformation available on the molecular interactionsed for the lipocalins, we suggest three testable hypothe-ut how Lazarillo could function in a signaling systemr axon guidance. First, the ligand that binds to the

hobic pocket could act as a guidance cue by inducing

Carpenter for generating mAb 10E6. This work was supported by NIHgrant to M. J. B. (NS25387). D. S. is the recipient of a NIH postdoc-toral fellowship (1F05TW04686-01), and M. D. G. holds a fellowshipfrom the Fulbright/MEC of Spain.

Accession numbers for Lazarillo sequence: GenBank U15656;EMBL Z38071.

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been (Accepted 30 September 1994)

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