Abstract
The extracellular portion of the macrophage mannose receptor, an endocytic receptor involved in clearance of glycoconjugates, contains eight domains related to the Ca(2+)-dependent carbohydrate-recognition domains (CRDs) of other C-type animal lectins. The characteristics of ligand binding to an expressed form of one of these CRDs (CRD-4) have been investigated. The expressed domain was found to be a monomer in solution. Results of a solid phase binding assay and a protease resistance assay show that CRD-4 of the mannose receptor undergoes a conformational rearrangement upon binding of Ca2+, correlating with its ability to bind sugar. CRD-4 requires two Ca2+ for sugar binding, even though sequence comparisons with other C-type CRDs suggested that it might bind only one Ca2+. The results are consistent with a ternary complex being formed between CRD-4, sugar, and Ca2+ as is seen in the crystal structure of the CRD of rat mannose-binding protein in complex with an oligosaccharide. The stability of Ca2+ binding is shown to be pH-dependent, a result that is pertinent to release of ligand by the receptor in the endosome. However, CRD-4 retains sugar binding activity at a lower pH than does the whole receptor, suggesting that the conformational change in this CRD alone may not be sufficient to allow release of ligand in the endosomes.
Highlights
The extracellular portionof the macrophage mannose The macrophagemannose receptor is amember of the C-type receptor, an endocytic receptor involvinedclearance of lectin family.C-type lectins, which bind carbohydrates in a glycocoqjugatesc,ontainseighdt omainsrelatedto
The characteris- 120 amino acids, which contain 14 invariant and 18 highly tics of ligand binding to an expressed form of one of conserved residues (1).Examples of C-type lectins are theasiathese CRDs (CRD-4) havebeeninvestigated.The ex- loglycoproteinreceptor, the selectins, and serum mannosepressed domain was found to abme onomer in solution. binding protein
The mannose receptor is unusual among CbrRctsgCoeoeeiRenqrsnssiDutudsuseeini-tdlsn4natdtsgncreeteenochrcqetofgaowufomtaiaeiritspCsstehosasalaamria2dytitsei+wgocrp,sohnonhhnstacwaoCforbsowiyretarihrncamboeod+ttlim haahnCtoatefdipRionotrniClrDnnlegyg-ax-ist4lbtyuoawrsopengesfaiiesaaetnCbahrrtgyiChrRlfaibeoaaDtniyrnznsgmm+sddeut.amieognnTda-genbhbn,poeeitrsen-uoevrdpteeeornsanesusucltetehgspao-rauttwCtps.yerhgyoRiipeehsltnyDehterepes-malerce(nipesFcctptheiihigdtxnoda.etsrsor1(amr5is)ecn)h.aEse.otiTilxdnhlwuhpe,anlearstaerfrtiseinhsbicptiareoototphrcnntttoeiooehonCfrcentptiRaiscocniDanortrastsyntibyops(ompine6hses,utIy71iIlond)tfrti.grerptAaapholtneefeessaiabrmnCteni,gRcnelNedmDaepin-sCnbttdoegreRirarinDmganinhceia(ttnvCpiCvaarsRliroi-tinDtctyoyegy-upiols4nesef-) tween CRD-4, sugar, and Ca2+ as is seen in the crystal can mimic the monosaccharide binding properties of the whole structure of theCRD of rat mannose-binding proteiinn receptor, but binds poorly to natural glycoproteins ( 6 )
Summary
The protein was removed and the plates washed with 1.25 M NaC1,25m~ Tris-HCI(pH7.8)(loadingbuffer)and blocked with loading buffer containing 5%(wh) BSA for 2 h at 4 "C. For Ca2+dependence assays, '"1-Man-BSA was added to wells containing appropriate concentrations of CaCl, and incubated at 4 "C for 2 h. The pH dependence of '251-Man-BSAbinding to mannose receptor fragments containing CRDs 4 and 5 and CRDs4-7 was determined using the same assay. These fragments were produced in insect cells as described previously(7).
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