Acyl-CoA-binding protein in the armadillo Harderian gland:its primary structure and possible role in lipid secretion

Brian M. Cavagnari a, Norma Sterin-Speziale a, Jorge M. A¡anni b, Jens Knudsen c,Jose A. Santome a;*

a Instituto de Qu|mica y Fisicoqu|mica Biologicas (IQUIFIB)-CONICET-UBA, Facultad de Farmacia y Bioqu|mica, UBA,Jun|n 956 (1113), Buenos Aires, Argentina

b Instituto de Neurociencia (INEUCI)-CONICET-UBA, Buenos Aires, Argentinac Institute of Biochemistry, University of Odense, DK-5230 Odense M, Denmark

Received 9 June 2000; received in revised form 31 October 2000; accepted 12 December 2000

Abstract

Similar to those of other species, the Harderian glands of armadillo produce an abundant lipid secretion, most of which iscomposed of 1-alkyl-2,3-diacylglycerol. Biosynthesis of this component is apparently performed with the participation of onecytosolic pool of acyl-CoA and another of free fatty acids. The acyl-CoA-binding protein (ACBP) is present at aconcentration at least 7-fold that of the heart-type fatty acid-binding protein (H-FABP), though lower than that in otherarmadillo organs such as liver and brain. The ACBP complete amino acid sequence was determined by Edman degradationof peptides generated by cleavage of the protein with cyanogen bromide, endopeptidase Glu-C, and trypsin. ACBP consistsof 86 residues and has a calculated molecular mass of 9783 Da, taking into account that an acetyl group is blocking the N-terminus. Identity percentages between armadillo Harderian gland ACBP and other known ACBPs show that the proteinbelongs to the liver-specific ACBP isoform (L-ACBP). The fact that the ACBP concentration is higher than that of FABPsuggests that the Harderian gland is able to store acyl-CoA amounts in ACBP larger than those of fatty acids in H-FABP for1-alkyl-2,3-diacylglycerol synthesis. ß 2001 Elsevier Science B.V. All rights reserved.

Keywords: Harderian gland; ACBP; Primary sequence; 1-alkyl-2,3-diacylglycerol

1. Introduction

The acyl-CoA-binding protein (ACBP) is an intra-cellular protein close to 10 kDa that binds medium-and long-chain acyl-CoA esters with high a¤nity but

is unable to do so with fatty acids [1]. It was discov-ered in bovine liver by Mogensen et al. in 1987 [2]and its amino acid sequence was determined thatsame year by Mikkelsen and Knudsen [3]. ACBPwas found by the above authors also in adipose tis-sue, intestinal mucosa, kidney, heart, brain, musclesand mammary gland. Is a highly conserved protein,76% of the sequence being identical when ACBPsfrom human, bovine, rat and pig proteins arecompared [1]. Taking into account the alignment ofthe 30 sequences published so far, ACBPs can bedivided into four groups: the liver-speci¢c isoform

0167-4838 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 8 3 8 ( 0 0 ) 0 0 2 9 4 - 6

Abbreviations: ACBP, acyl-CoA-binding protein; ADG, 1-al-kyl-2,3-diacylglycerol ; CNBr, cyanogen bromide; DBI, diaze-pam-binding inhibitor; DTT, dithiothreitol ; FABP, fatty acid-binding protein

* Corresponding author. Fax: +54-11-4508-3652;E-mail : santome@qb.¡yb.uba.ar

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(L-ACBP), the testis-speci¢c isoform (T-ACBP), thebrain-speci¢c isoform (B-ACBP) and the membrane-bound isoform (M-ACBP). L-ACBP is the generallyexpressed ACBP isoform [4].

It has been suggested that ACBP acts as an intra-cellular carrier of acyl-CoA thus preventing its deter-gent e¡ect on membranes as well as its inhibitorye¡ects on some lipid metabolism enzymes [5].

Although the highest concentration of ACBP wasreported in liver [6], the amount of ACBP found inbovine and rat liver is only 3.3 and 6.4 Wg/mg ofsoluble protein, respectively [1], while that of fattyacid-binding proteins (FABPs) in mammalian liveris about 50 Wg/mg [7].

We recently reported the presence of an FABP in aHarderian gland for the ¢rst time, and determined itspartial amino acid sequence for the armadillo gland[8]. These orbital glands were ¢rst described by J.J.Harder in 1694 and are present, with few exceptions,in all terrestrial vertebrates [9,10]. They are exocrineglands that secrete lipids through a merocrine mech-anism [11]. 1-Alkyl-2,3-diacylglycerol (ADG) is themajor lipid component in mouse [12], guinea pig[13], rabbit [14], and golden hamster [15] Harderianglands, but alkyl and acyl positions of such lipid inthese animals are species-speci¢c [11]. Secretory lipidsaccount for approx. 20% of Harderian gland freshtissue weight and are secreted in large amounts[16]. Lipids secreted by Harderian glands are ex-tremely important and a great deal of functionsare ascribed to them. For instance, they act as alubricant facilitating eyelid movement, they act as asolvent for biologically active substances like phero-mones or growth factors, they have a thermoregulat-ing function, and a bactericidal e¡ect has also beenattributed to them [16]. Unexpectedly, despite its im-portant lipid metabolism, this gland has only verysmall amounts of FABP [8]. In this work we foundthat there is an ACBP in the Harderian gland andthat its concentration is far higher than that of theH-FABP also present in the gland. We determined itscomplete amino acid sequence and also that ADG isthe main secretory product of the armadillo Harder-ian gland. The convenience of having high amountsof ACBP in comparison with those of FABP for theHarderian gland lipid secreting function is herein dis-cussed.

2. Materials and methods

2.1. Materials

Harderian glands were obtained from adult arma-dillos of the Chaetophractus villosus species. Theseanimals were fed Purina dog chow and water adlibitum. They were kept at 23³C under a 12^12 hlight-darkness cycle in the special breeding roomsof the INEUCI.

Silica gel G plates and standards were obtainedfrom Sigma (St. Louis, MO, USA). [1-14C]Palmi-toyl-CoA (57.1 Ci/mol) was from Du Pont/NewEngland Nuclear (Boston, MA, USA). SephadexG-75, DTT, EDTA and tricine were purchasedfrom Sigma. Acrylamide, N,NP-methylene-bis-acryl-amide, 2-mercaptoethanol and Coomassie blue werefrom Bio-Rad (Richmond, CA, USA). Reagents foramino acid sequencing were obtained from AppliedBiosystems (Foster City, CA, USA). All other re-agents were of analytical grade.

2.2. Preparation of 106 000Ug supernatants

Harderian gland and other tissue assayed pieceswere suspended separately (1:1) in 20 mM sodiumphosphate (pH 7.4), 150 mM KCl, 1 mM EDTA,1 mM dithiothreitol, and homogenized in a glass-Te£on homogenizer. The homogenates were thencentrifuged at 30 000Ug at 4³C for 15 min. The re-sulting supernatants were further ultracentrifuged at106 000Ug at 4³C for 90 min.

2.3. Coelution of cytosolic proteins with radiolabeledpalmitoyl-CoA

The 106 000Ug Harderian gland supernatant (0.5ml) was incubated with 0.5 WCi of [1-14C]palmitoyl-CoA during 20 min at 20³C. The mixture was thenloaded to a Sephadex G-75 column (0.85U46 cm)equilibrated with 30 mM Tris-HCl, pH 8.5. Elutionwas performed at 4³C with the same bu¡er at a £owrate of 2 ml/h. Fractions of 0.7 ml were collected andassayed for radioactivity in an Allac 1214 RackbetaLiquid Scintillation Counter.

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2.4. ELISA of ACBP

ACBP was assayed by a multiple-layer enzyme-linked immunochemical procedure as described byPawlak and Smith [17], except for the reaction ofACBP with immobilized rabbit anti-ACBP whichwas carried out in 10 mM Tris-HCl (pH 9.0), 0.5M NaCl, 2.6 mM KCl, 0.1% bovine serum albumin,0.02% NaN3, overnight at 4³C. The layers, sequen-tially attached to a 96-well plastic plate, consistedsuccessively of monospeci¢c rabbit anti-ACBP IgG,ACBP antigen, biotinylated rabbit anti-ACBP IgG,avidin and biotinylated alkaline phosphatase; thechromogenic substrate was p-nitrophenyl phosphate.

Production and puri¢cation of anti-ACBP anti-bodies were performed as per Knudsen et al. [18].

2.5. Protein determination

The protein content in di¡erent armadillo organswas measured according to Lowry et al. [19].

2.6. Puri¢cation of the Harderian gland ACBP

The clear high-speed supernatant was applied to aSephadex G-75 column (2.5U40 cm), equilibratedwith 30 mM Tris-HCl, pH 8.5, at 4³C. Elution wasperformed with the same bu¡er at a £ow rate of 18ml/h. Pooled fractions containing ACBP (elution vol-ume 120^140 ml) were concentrated and desalted us-ing a Centriprep concentrator (Amicon), and chro-matographed on an FPLC system (Pharmacia,Biotech) with a Mono-Q column equilibrated withthe same bu¡er. Proteins were eluted at a £ow rateof 1 ml/min utilizing a 50 ml linear gradient of 0.1 MKCl in the starting bu¡er. Proteins present in theACBP-containing fraction were separated by HPLCon a Brownlee C8 RP-300 (30U2.1 mm) column,equilibrated with 5% solvent B (80% acetonitrileand 0.08% TFA). Elution was performed at a £owrate of 200 Wl/min, using 5% solvent B for 5 min anda linear gradient of 5^90% solvent B for 55 min. Thematerial of the ACBP peak was immediately storedat 320³C.

2.7. CNBr-iodide cleavage

Cleavage of both methionyl and tryptophanyl pep-

tide bonds in ACBP was performed by the method ofHuang and Huang [20] according to the followingprocedure. Approx. 100 Wg of the puri¢ed proteinwas treated in an Eppendorf tube with 40 Wl ofCNBr (8 Wg/Wl in 70% formic acid) and 4 Wl of 12mM NaI in water and kept in the dark at roomtemperature for 24 h. The liquid was completelyevaporated in a Speed-Vac centrifuge, and 2 vols.of 150 Wl were added and evaporated each time. Pep-tides were fractionated by HPLC on a Brownlee C4

(100U2.1 mm) column, equilibrated with solvent A(0.1% TFA). Elution was performed at a £ow rate of150 Wl/min, using solvent A for 5 min, and threelinear gradients of solvent B (80% acetonitrile and0.08% TFA): 0^10% for 5 min, 10^60% for 60 minand 60^90% for 5 min. Fractions 1^4 were submittedto Edman degradation.

2.8. Digestion with endopeptidase Glu-C

Puri¢ed ACBP (approx. 40 Wg) was dissolved in 70Wl of 8 M urea and incubated at 20³C during 10 min.The sample was diluted with 67.5 Wl of 200 mM Tris-HCl, pH 7.9, and then with 5 Wl of endoproteinaseGlu-C (0.5 Wg/Wl). After incubation at 20³C during24 h, the sample was acidi¢ed with 1.5 Wl of TFA.The reaction products were separated by RP-HPLCon a Vydac C18 (220U2.1 mm) column, equilibratedwith solvent A (0.1% TFA). Elution was performedat a £ow rate of 175 Wl/min, using solvent A for 5 minand three linear gradients of solvent B (80% acetoni-trile and 0.08% TFA): 0^10% for 3 min, 10^80% for60 min and 80^90% during 4 min. Fractions 2 and 6were subjected to Edman degradation.

2.9. Electrophoresis

SDS-PAGE was performed as described by Schag-ger and von Jagow [21] using a Mini-Protean II de-vice (Bio-Rad). Total monomer concentration was16% and no spacer gel was utilized. Gels werescanned and then analyzed with a Jandel SigmaScan 2.0.

2.10. In-gel digestion of the Harderian gland ACBPfor internal sequence analysis

The ACBP-containing fraction from the Sephadex

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G-75 column was submitted to SDS-PAGE. The gelwas stained with Coomassie blue. The ACBP bandwas excised and subjected to in-gel digestion withtrypsin following the procedure of Rosenfeld et al.[22] as modi¢ed by Hellman et al. [23]. The peptidemixture was separated on a Brownlee C18 column(220U2.1 mm) equilibrated with TFA 0.1%. Elutionwas performed at a £ow rate of 150 Wl/min with a 0^90% acetonitrile (80%) and TFA (0.08%) gradient for75 min. Fraction 5 was submitted to Edman degra-dation.

2.11. Deblocking of the N-terminus

The puri¢ed protein was blotted onto a PVDFmembrane. The immobilized protein was placed ina small tube enclosed in another tube containing an-hydrous TFA, the latter being sealed and heated at60³C for 60 min to expose the membrane to TFAvapor, as per Tsunasawa and Hirano [24].

2.12. Amino acid sequencing

Amino acid sequencing was carried out on an Ap-plied Biosystems 477A protein sequencer equippedwith an on-line 120A phenylthiohydantoin analyzer.

2.13. Extraction of fatty acids and acyl-CoA esters

Acyl-CoA esters were isolated as described byRosendal and Knudsen [25]. Harderian glands wereextracted from armadillos, weighed, readily frozenand stored at 370³C until treated the followingday. Ice-cold chloroform-methanol 2:1 (v/v) wasadded, followed by water to give a total chloro-form-methanol-water ratio of 5:2.5:2. All the re-maining steps were performed at 4³C. The glandswere homogenized using heptadecanoyl-CoA andheptanoic acid as internal standards. Separation ofthe two phases was facilitated by centrifugation at3200Ug during 45 min. The upper phase containingthe acyl-CoA esters was removed. The interphaseprotein and the lower phase were extracted threetimes with methanol-2 M ammonium acetate 4:1(v/v). The three extracts were combined with theupper phase from the chloroform-methanol-water ex-traction and lyophilized (aqueous extract). The lower

chloroform phase, corresponding to fatty acids, wasdried under a £ow of nitrogen (organic extract).

Both aqueous and organic extracts were submittedto transmethylation to determine the fatty acid pro-¢le and concentration.

2.14. Harderian gland secretion lipids

The Harderian gland secretion lipids were twiceextracted with 5 ml of chloroform-methanol 2:1(v/v) at 20³C and the solvent was then evaporatedunder a nitrogen £ow. The resulting residue was dis-solved in 50 Wl chloroform and applied to precoatedsilica gel G plates, and developed in n-hexane-diethylester-acetic acid 80:20:1 (v/v). A small portion wasrun in parallel so as to detect it under iodine vapor.After isolation, the ADGs were scraped o¡ for meth-yl ester obtention.

2.15. Preparation of fatty acid methyl esters

Each zone of the thin-layer plate was transesteri-¢ed with 1 ml of sulfuric acid 2% (v/v) in methanol ina dry shaker at 60³C for 2 h. Samples were ¢ltered toseparate the silica. Another milliliter of sulfuric acid-

Fig. 1. Coelution of armadillo Harderian gland cytosolic pro-teins with [1-14C]palmitoyl-CoA. The 106 000Ug supernatantwas incubated with [1-14C]palmitoyl-CoA and then fractionatedon a Sephadex G-75 column equilibrated with 30 mM Tris-HCl, pH 8.5. Eluted fractions were assayed for radioactivity.The ACBP peak is indicated by an arrow.

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methanol was added in order to improve sample re-covery. 4 ml chloroform and 1.4 ml water wereadded and the samples vortexed vigorously. Aftercentrifugation at 1000 rpm for 5 min, the upperphase was separated and washed twice with 1.4 mlof water. The organic phase was dried under a nitro-gen stream and samples were frozen at 320³C.

2.16. Fatty acid determination

Gas chromatography of the fatty acid methyl es-ters was carried out in a gas chromatograph (SeriesGC-8A) equipped with a £ame ionization detectorand a 30 mU0.25 mm DR-23 capillary column at140³C. Nitrogen was used as the carrier gas at apressure of 6 kg/cm2, and the percentage composi-tion was calculated by means of an automatic inte-grator [26]. The peaks in the chromatogram wereidenti¢ed by comparing retention times with knownfatty acid methyl ester Sigma standards.

Fig. 3. ACBP content in the cytosolic fraction of di¡erent armadillo organs. Cytosolic fractions from various armadillo tissues wereobtained and analyzed by ELISA as described in Section 2. The results are means of duplicates þ half the di¡erence.

Fig. 2. 16% SDS-PAGE analysis [21] of the ACBP-containingfraction from the Sephadex G-75 column. Protein bands werestained with Coomassie blue. ACBP and FABP bands, identi-¢ed by their amino acid sequence, are indicated by arrows.

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3. Results

3.1. Search for a Harderian gland ACBP

The Harderian gland cytosolic fraction, obtainedby ultracentrifugation at 106 000Ug, was incubatedwith [1-14C]palmitoyl-CoA and fractionated on theSephadex G-75 column used for coelution. Two ma-jor radioactive peaks were obtained (Fig. 1): the ¢rst^ corresponding to a molecular mass over 60 kDa ^may re£ect the presence of contaminating serum al-bumin; the second corresponds to the molecularmass proteins of FABPs and ACBPs. A fraction ofthe material of this peak was submitted to SDS-PAGE. Fig. 2 shows the heart-type FABP band re-ported by Cavagnari et al. in 1998 [8], and a bandcorresponding approximately to a 10 kDa protein.The partial amino acid sequence of the latter showsa high identity degree with that of previously re-ported ACBPs. A computer image measurementanalysis of the scanned gel indicated that ACBP ispresent at a concentration at least 7-fold that ofheart-type FABP.

3.2. ACBP content in di¡erent armadillo organs

Taking into account the above ACBP/FABP ratio,we investigated if the ACBP is present at a higherconcentration in Harderian glands than in other or-gans. The content of ACBP in various armadillo or-gans was determined by quantitative ELISA.

As shown in Fig. 3, the highest ACBP concentra-tion is present in liver, followed by ovary and brain.The other organs tested have a signi¢cantly lowerACBP concentration. This agrees with previous re-sults for rat and cow [3,6].

This ¢nding indicates that though ACBP is not avery abundant protein in the Harderian gland ^ atleast when compared with the ACBP content in liverand brain ^ the ACBP/FABP ratio is signi¢cantlyhigher in this organ.

3.3. Puri¢cation of ACBP

The Sephadex G-75 fraction containing ACBP waschromatographed on a Mono-Q column. Two peakswere obtained (Fig. 4A). One of them eluted in thepass-through fraction and the second at the begin-

ning of the KCl gradient. The latter was concen-trated and then submitted to SDS-PAGE (Fig. 5,lane 1). Two bands were obtained: a lightly coloredband, with a position corresponding to that of theFABP molecular mass [8], and a strongly colored onecorresponding to that of the ACBP. Puri¢cation ofthe armadillo Harderian gland ACBP was ¢nally ob-tained by subjecting the material of peak 2 (Fig. 4A)

Fig. 4. (A) Anion exchange chromatography. The ACBP-con-taining fraction obtained by Sephadex G-75 chromatographywas loaded to a Mono-Q column previously equilibrated withTris-HCl 30 mM, pH 8.5. Proteins were eluted with a discon-tinuous gradient of KCl. The ACBP-containing peak is indi-cated by an arrow. (B) RP-HPLC separation of the proteinspresent in the Mono-Q fraction containing ACBP. This fractionwas loaded to a Brownlee C8 RP-300 (30U2.1 mm) columnequilibrated with 5% solvent B (80% acetonitrile and 0.08%TFA). Elution was performed at a £ow rate of 200 Wl/min, us-ing 5% solvent B for 5 min and a linear gradient of 5^90% sol-vent B during 55 min. The ACBP-containing peak is indicatedby an arrow.

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to reverse-phase HPLC, thus separating the two pro-teins (Fig. 4B). The ACBP was homogeneous inSDS-PAGE and calculated to have an apparent mo-lecular mass of 10 kDa (Fig. 5, lane 3).

3.4. Primary structure

Two small samples were separated from theACBP-containing fraction eluted from the prepara-tive Sephadex G-75 column and then concentratedand submitted to SDS-PAGE (Fig. 2). The ACBP

band from one of the above was digested by the`in-gel' procedure of Rosenfeld et al. [22] and theresulting peptides were fractionated by reverse-phaseHPLC (Fig. 6A) and stored for later sequencing. TheACBP band from the other sample was blotted ontoa PVDF membrane and subjected to sequencing inan automatic sequencer. Attempts to obtain theN-terminal sequence were unsuccessful, thus suggest-

C

Fig. 6. (A) HPLC fractionation of peptides generated by `in-gel'digestion of the armadillo Harderian gland ACBP band. Thepeptide mixture was separated on a C18 reverse-phase columnequilibrated with 0.1% (v/v) TFA in water, and eluted with agradient of 80% (v/v) acetonitrile and 0.08% (v/v) TFA. Peak 5was submitted to Edman degradation. (B) HPLC fractionationof peptides generated by CNBr-iodide cleavage of the glandACBP. The peptide mixture was separated on a C4 reverse-phase column equilibrated with 0.1% (v/v) TFA in water, andeluted with three linear gradients of 80% (v/v) acetonitrile and0.08% (v/v) TFA. Peaks 1^4 were subjected to Edman degrada-tion. (C) HPLC fractionation of peptides generated by cleavageof the armadillo Harderian gland ACBP with endopeptidaseGlu-C. The peptide mixture was separated on a C18 reverse-phase column equilibrated with 0.1% (v/v) TFA in water, andeluted with three linear gradients of 80% (v/v) acetonitrile and0.08% (v/v) TFA. Materials from peaks 2 and 6 were submittedto Edman degradation.

Fig. 5. 16% SDS-PAGE analysis [21] of ACBP-containing frac-tions. Lane 1, material obtained by anion exchange chromatog-raphy on a Mono-Q column; lane 3, ACBP obtained by RP-HPLC puri¢cation of the preceding fraction. Protein bandswere stained with Coomassie blue. Lane 2, void.

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ing that the ACBP has a blocked N-terminus. Theblocking group was removed by treatment of thePVDF band with gaseous TFA as this method hasproven to be e¡ective only on proteins having acetyl-serine/threonine as happens in most ACBPs. Whensaid band was subjected to sequencing, the peptideobtained was SQAEFDKAAEEVKNL.

In order to determine the complete primary struc-ture, the ACBP obtained after the last puri¢cationstep was divided into two fractions. One of them wassubmitted to chemical cleavage with CNBr-iodide.Peptides were fractionated by reverse-phase HPLC(Fig. 6B). The peaks were numbered from 1 to 4.Although the peptide fragments were not well re-solved, they were submitted to Edman degradationtaking into account that there was not enough pep-tide mass for rechromatography and that armadilloHarderian glands are di¤cult to obtain. The materialcorresponding to peaks 1 and 2 showed the sequenceLFIYSHYKQATVGDINTERPGXX(D) which wasalso present ^ in very small amounts ^ in the materialfrom peaks 3 and 4; that from peak 3 was KSYIDK-VEELKKKYGI. The sequence of the material cor-

responding to peak 4 agrees with the presence ofpeptide NQLKGTSKEDAMKSYIDKV.

The puri¢ed protein of the other fraction was di-gested with endopeptidase Glu-C and the resultingpeptides were fractionated by reverse-phase HPLC(Fig. 6C). The material corresponding to peaks 2and 6 was selected for Edman degradation basedon the information provided by a diode array sys-tem. Finally, in order to obtain the complete primarystructure, we looked for the tryptic fragmentTKPADDEMLFIYSHYK in the `in-gel'-digestedprotein and, by means of the diode array system,we found it in the material of peak 5 due to its ty-rosine content (Fig. 6A).

Harderian gland ACBP is 86 residues long andcontains no cysteines. Considering this and the iden-tity percentages shown in Fig. 7, it may be concludedthat the Harderian gland ACBP is of a liver-speci¢cACBP type.

As calculated from its amino acid sequence andtaking into account that the acetyl group blocks itsN-terminus, the armadillo Harderian gland ACBPhas a molecular mass of 9783 Da.

Fig. 7. Sequence alignment of peptides derived from armadillo Harderian gland ACBP with the corresponding fragments of variousACBPs obtained from the SWISS-PROT data bank. Identity percentages between armadillo Harderian gland ACBP and the corre-sponding segments of the other ACBPs are indicated. Full arrows represent the segments of the sequence of peptides generated bycleavage of the armadillo Harderian gland ACBP with CNBr (B), endopeptidase Glu-C (G) and trypsin (T) respectively. H representsthe segment of the sequence of the peptide generated by deblocking the ACBP with TFA vapor. Peptide alphanumeric symbols areconsistent with those of Fig. 6. ARMAD, armadillo. Amino acid residues identical with those in ARMAD ACBP are shown in boldtype.

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3.5. Harderian gland secretion lipids

In an attempt to ¢nd some relationship betweenthe high ACBP/FABP ratio and the Harderian glandlipid metabolism, we determined the composition ofits secretory lipids. When Harderian gland lipidswere separated by preparative TLC (not shown), agreat predominance of ADGs (about 90%) was ob-served ^ free cholesterol also being present in smallamounts. The same as in mouse [12], guinea pig [27]and golden hamster [15], ADGs are the main secre-tion product of the armadillo Harderian gland.

After isolation, the gland ADGs were scraped o¡for methyl ester obtention and fatty acid determina-tion in the ester (Fig. 8). It contains 16:0 (22%), 18:1(21.4%) and 18:0 (18.6%) as major components, fol-lowed by 18:2 (8.5%). In addition, we found even-and odd-numbered straight-chain fatty acids, rangingfrom 14 to 23, and also branched fatty acids (15:0) insmall amounts. In the case of 18:1, two isomers werefound, n = 9 (19.5%) and n = 7 (1.9%). The same hap-pened with 16:1 (n = 9 and n = 7, 2% each). 3.6. Quantitation and content of free cytosolic fatty

acids and acyl-CoA esters

The predominant cytosolic free fatty acid obtainedfrom female armadillo Harderian gland is 18:1(31.4%), followed by 18:0 (16.9%), 18:2 (13.5%)

Fig. 9. Fatty acid cytosolic pool of the armadillo Harderiangland. Cytosolic fatty acids isolated as per Rosendal and Knud-sen [25] were determined by gas chromatography. The data arepresented as the means þ S.E.M from three separate experimentswith each determination carried out in duplicate.

Fig. 8. Fatty acid composition of Harderian gland ADGs.These lipids were scraped o¡ from silica gel G plates and theirconstituent fatty acids were determined by gas chromatography.The data are presented as the means þ S.E.M from three sepa-rate experiments with each determination carried out in dupli-cate. br, branched fatty acid.

Fig. 10. Acyl-CoA ester cytosolic pool of the armadillo Harder-ian gland. Cytosolic acyl-CoA esters isolated as described byRosendal and Knudsen [25] were determined by gas chromatog-raphy. The data are presented as the means þ S.E.M from threeseparate experiments with each determination carried out in du-plicate.

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and 16:0 (9.3%), whereas other fatty acids arepresent at a concentration under 5%. Only even-numbered fatty acids were found, ranging from 14to 24, both saturated and unsaturated as shown inFig. 9.

On the other hand, the predominant fatty acidfound in cytosolic acyl-CoA esters is 18:0 (26.1%),followed by 18:1 (21.8%), 16:0 (16.1%) and 18:2(10.8%). Except for 22:2 (7.5%), all the other acyl-CoA esters were present at a concentration under5%. Only even-numbered esters were found, rangingfrom 14 to 22, both saturated and unsaturated asshown in Fig. 10.

4. Discussion

Both an ACBP and a heart-type FABP are presentin armadillo Harderian glands. Previously reportedresults [8] propose that armadillo Harderian glandshave only one FABP at a low concentration despitethe high lipid content and the diversity of lipid met-abolic pathways reported for the Harderian glands ofspecies studied so far [16,28]. The low concentrationof FABP in this gland [8] suggests that it might con-tain a higher concentration of other fatty acid/acyl-CoA-binding proteins. Our ¢nding of ACBP at ahigh concentration in this gland supports our pre-vious hypothesis.

In Harderian glands, fatty acids are synthesized incytosol by two enzymes: acetyl-CoA carboxylase andfatty acid synthetase [16]. Palmitic acid is usually themain product of fatty acid synthetase in Harderianglands, but several other fatty acids ^ includingmethyl-branched fatty acids ^ are also produced[16]. Two di¡erent long-chain acyl-CoA synthetaseswere identi¢ed in the Harderian gland of guinea pigsby Kono et al. in 1996 [29]. One was located inmicrosomes and the other in the mitochondria. Sub-strate speci¢city and catalytic activity of the Harder-ian gland mitochondrial enzyme for palmitic, linoleicand arachidonic acids are similar to those of thecorresponding liver enzyme, but the catalytic andimmunogenic characteristics of the microsomal syn-thetase are di¡erent from those of microsomes andthe mitochondria of the guinea pig liver. Taking intoaccount the large Vmax value of the Harderian glandmicrosomal acyl-CoA synthetase, Kono et al. [29]

suggest that it contributes to the synthesis of a largeamount of secretory lipids and that its high Km valuedoes so due to the existence of large amounts ofcellular lipids in this organ. In the presence of ace-tyl-CoA, propionyl-CoA, malonyl-CoA and methyl-malonyl-CoA, the enzyme from guinea pigs can yieldthe branched fatty acids needed to synthesize theADG present in lipids secreted by Harderian glandsof already studied animals [16]. According to thesestudies, the acyl-CoA used for triglyceride and ADGsynthesis comes mainly from glycolysis (Fig. 11).

We show herein that ADG is the main secretoryproduct in the armadillo Harderian gland as it is inother species studied. It is interesting that, althoughfatty acid chain lengths in the free fatty acid andacyl-CoA pools are very similar, the degrees of de-saturation are very di¡erent; this may be due to thespeci¢city of the acyl-CoA synthetase in the gland.

The fact that the ratio of the four predominantacyl-CoA esters (18:0, 18:1, 16:0 and 18:2) is thesame and approximately that of the fatty acidspresent in ADGs (16:0, 18:1, 18:0 and 18:2), indi-cates that acyl-CoA bound to ACBP may act as apool for the ADG synthesis pathway.

Fig. 11. Metabolic pathway of Harderian gland secretion lipids.Synthesis of ADG and triacylglycerol originated by glycolysis.The FABP and ACBP importance in leading to a pool of freefatty acids and acyl-CoA respectively is emphasized. FA, fattyacids.

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The presence of ACBP in the armadillo Harderiangland may allow accumulation of CoA esters withoutinhibition of the acyl-CoA synthetase supporting thesynthesis of neutral lipids. Previous work has indeedshown that the acyl-CoA/ACBP complex can conveyacyl-CoA to microsomes for the synthesis of glycero-lipids [30].

The most important secretion products of the ar-madillo Harderian gland are ADGs as estimated byTLC. They are secreted by a merocrine mechanismand contribute to facilitate eyelid movement. In ad-dition to having a bactericidal e¡ect, they act as asolvent of bioactive substances and play an impor-tant role in thermoregulation.

The Harderian gland and the adipose tissue sharethe capability of synthesizing acylglycerols. However,while the adipose tissue releases fatty acids into theblood so that they may be used in other tissues,Harderian glands secrete large amounts of acylgly-cerols. This could be the reason why a high concen-tration of adipose tissue FABP is required so as tobind the fatty acids to be delivered to other tissues ^this not occurring in neutral lipid-secreting Harder-ian glands. In this gland, the high ACBP/FABP ratiomay be important for the existence of said cytosolicpool of acyl-CoA esters which is available to supportthe ADG synthesis pathway of Fig. 11, which showsthat the pool of acyl-CoA bound to ACBP plays amore important role than that of fatty acids boundto FABP.

Harderian gland ACBP may be capable of havingthe following e¡ects: (a) maintaining a large pool ofthe acyl-CoA esters synthesized by the microsomallong-chain acyl-CoA synthetase; these esters, boundto ACBP, are protected from acyl-CoA hydrolasesand have no detergent e¡ects on membranes; (b)the acyl-CoA synthesis may increase due to the pres-ence of ACBP, taking into account that long-chainacyl-CoA synthesis is stimulated by removing long-chain acyl-CoA esters [31]; (c) increasing acyl-CoAsynthesis by stimulation of acetyl-CoA carboxylaseconsidering that the activity of this enzyme is re-stored by ACBP in rat liver when inhibited bylong-chain acyl-CoA esters [31]; (d) increasing thesynthesis of the acylglycerol derivatives secreted bythe gland in view of the acyl-CoA sequestered byACBP which can be delivered to the correspondingsynthetases.

Acknowledgements

We are greatly indebted to Dr. Emir Speziale forperforming lipid analyses. Amino acid sequence anal-ysis was performed at the LANAIS-PRO (NationalProtein Sequencing Facility, UBA-CONICET, Bue-nos Aires, Argentina) and we wish to thank S.B.Linskens and E.V. Dacci for their skilful technicalwork. This work was supported by grants from theUniversidad de Buenos Aires and CONICET.

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