Abstract

An acidic glycoconjugate containing mannose, galactose and phosphate in approximately equimolar amounts was extracted from Leishmania donovani promastigotes and partially characterized. The glycoconjugate could be metabolically labeled with either [3H]mannose or [3H]galactose and was extractable from a delipidated residue fraction with water/ethanol/diethyl ether/pyridine/concentrated NH4OH (15:15:5:1:0.017) at 25 degrees C. The radioactively labeled glycoconjugate was found to possess the following characteristics: 1) comprised 45-60% of the total [3H]mannose label incorporated into macromolecules; 2) was soluble in alkaline solvents and 0.5% Triton X-100; 3) migrated as a broad band upon electrophoresis on sodium dodecyl sulfate-polyacrylamide gels with an approximate molecular weight of 15,000-30,000; 4) bound to DE52 cellulose and was eluted with a salt gradient of 0-0.1 M NaCl; 5) was insensitive to Pronase, hyaluronidase, chondroitinase, endo-beta-N-acetylglucosaminidase H, and endo-beta-galactosidase; and 6) possessed hydrophobic properties. An unusual feature of the glycoconjugate was its lability to mild acid hydrolysis (0.02 N HCl, 15 min, 60 degrees C). As determined by alkaline phosphatase and glycosidase digestion and paper chromatographic analysis, the major fragment generated by mild acid hydrolysis was found to be a phosphorylated galactosyl-beta-mannose disaccharide. All of these characteristics suggest that the glycoconjugate may be a polysaccharide and, possibly, may be important in parasite-host cell interactions.

Highlights

  • An acidic glycoconjugate containing mannogsea,lac- which bind to lectins, as well as entities that tosaenpdhosphatiaenpproximateleyquimolar confer a negative charge to these structures

  • Possessed hydrophobic properties.An unusual feature Materials-All materials were obtained as follows: Dulbecco’s modof the glycoconjugate was its lability to mild acid hy- ified Eagle’s medium (DME 430-2100) and fetal calf serum from drolysis (0.02N HCI, 15 min, 6 0 “C).As determined by Gibco; [2-3H]mannose (18.4Ci/mmol) and [6-3H]galactose(9.1 Ci/

  • Alkaline phosphatase andglycosidasedigestion and pa- mmol) from New England Nuclear; [35S]methionine(1300 Ci/mmol) perchromatographic analysis, the majorfragment generated by mild acid hydrolysis was found to be a phosphorylated galactosyl-@-mannosedisaccharide

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Summary

Expression of an Unusual Acidic Glycoconjugate in Leishmania donovani”

An acidic glycoconjugate containing mannogsea,lac- which bind to lectins, as well as entities that tosaenpdhosphatiaenpproximateleyquimolar confer a negative charge to these structures. Using antisera amounts was extracted from Leishmaniadonovani against whole organisms, a number of investigators working promastigotes andpartially characterized. Theglyco- with several distinct human leishmanial species have reported conjugate could be metabolically labeled with either that the promastigote forms release antigenically active gly-. [3H]mannoseor[3H]galactoseand was extractable coproteins into culture media [3,4,5,6]. Shiro et al [7] have shown by immunoprecipitation techniques ethanolldiethyl etherlpyridlnelconcentrated NH,OH with antisera against purified L. donovani promastigote cell wcomg(11hnl0aya5a0scn:rs;o1noas3c5odoco):ltsiu5nuemer:bjmla1uiilgsbe:gtr0ediaiac.lnot0itsende:1adce71wloca))kryaascaplsotlaoims2nrfuoba5eplurtfre“aosnidoCtasdeeld.v-dpTteobio4nhnal5teytpns-oa6rdoaca0smrnsdu%ydeaiplsoacos0oarmn.foct5itmhtde% ihgveleoeeeelTcllfesttyocrrowluilotltaaolpoiebtlwnhshe[;’oilHXaner2dnIeg-)siscsgtohufaairWrtsafebaspoecmaheohpyafapejdovtrrrh,ereawpetgbeaelp-erycrargceootuoipmnnocotnaaorasstinnttjbithiughinegooagactitetsheaoetfnhlcmoatoetriinigmpcoteaananlroiasanafs.nininLdatgeelysipsasphainsrmrotaoaidpfanupcltichraheoaexdreaoixgmcntlrtyoaeacutrcoeaiezcnleaoliu.qtnilujoIauinnr--. An unusual feature of the glycoconjugate is its lability to mild acid hygradient of 0-0.1 M NaCl; 5) was insensitive to Pro- drolysis. Nase, hyaluronidase, chondroitinase,endo-@-N-acetylglucosaminidase H, and endo-&galactosidase; and 6)

EXPERIMENTAL PROCEDURES
Characterization of the Radioactive Glycoconjugate
Temperature exPterraccteanbtle
FRACTION NUMBER
Man azzm
Analysis of Unlabeled Glycoconjugate
DISCUSSION

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