Abstract
cDNA comprising the entire length of the rat Kupffer cell receptor (Mr = 88,000 and 77,000) for carbohydrates with an affinity for fucose and galactose was isolated, and its nucleotide sequence was determined. Receptor cDNA encoded a protein containing 550 amino acid residues with a Mr = 61,104. This Mr was consistent with the size of the deglycosylated receptor which was found to contain two polypeptides by gel electrophoresis with Mr = 58,000 and 52,000, respectively. Edman degradation of the receptor yielded a sequence which corresponded to amino acid residues 83-104 of the sequence derived from the cDNA. This confirmed that the cDNA which had been isolated corresponded to mRNA for the receptor and suggested that the smaller polypeptide in receptor preparations arises by proteolysis of the intact receptor. Amino acid composition of the receptor was nearly identical to that predicted by the cDNA. The Kupffer cell receptor was found to be homologous to other carbohydrate binding proteins including the hepatic receptors with different binding specificities. The Kupffer cell receptor also contained a series of 18 contiguous, homologous sequences with an average length of 14 residues.
Highlights
CDNA comprising theentirelengthof the rat Kupfferand monocytes examined, except Kupffer cells, and has not cell receptor (Mr = 88,000 and 77,000) for carbohy- been found in any tissue besides liver (7)
The following materials were obtained commercially: containeda series of 18 contiguous,homologous se- DNA restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase quences with an average length of 14 residues
Since the receptor was a glycoprotein and may not migrate normally on gel electrophoresis, it was deglycosylated with N-glycanase or a mixture of glycosidases and sub
Summary
CDNA comprising theentirelengthof the rat Kupfferand monocytes examined, except Kupffer cells, and has not cell receptor (Mr = 88,000 and 77,000) for carbohy- been found in any tissue besides liver (7). The drates with an affinity for fucose and galactose was receptor binds glycoproteins with nonreducing terminal fuisolated, and its nucleotide sequencewas determined. The derived protein sequencereveals thatthe Kupffer cell receptor belongs to a family of proteins with related carbohydrate binding sites (8). EXPERIMENTALPROCEDURES confirmed that the cDNA which had been isolated corM- aterials-Kupffer cell receptor (5), anti-receptor antibodies (5), tcraheorsamipsteoptsnohbdseyietsdiompntraoololtmeefroRltpyNhoseAilsyrpfoeoecfrpettpthihtdweoeerraeiiscnnnetpreaeatcocrtrelyparentociddersepupnAtgrotmgiercep.iasnaltoreadtoaticoitndhsatSwdaidoteBargreseilpeoyp,t-coSfrouceesopciadocarcarcseiushddesa3Dpas6nes0e,(0dun9eme)ss,uCycornrlnaoitimbhsoeeteirdsneiidienzdiadeuarorsml-ieweapri-(e.tlNhrOof-r)rali,iencgageaogtennenydunlslctZgaslae6-sNloIu-at-lpicaadptcbeloeesiteswlyedaeldmpgbrireayonlatmitechdiataneodassemAneaudmas(ne1iinunn1g---). The Xgtll oligo(dT)-primed rat livercDNA library and found to be homologous to other carbohydrate bindingEcoRI methylase were gifts from Dr James Paulson (UCLA Medical proteins including the hepatic receptowrsith different Center) and Dr Paul Modrich (Duke University Medical Center), binding specificities. (Bethesda Research Laboratories or Pharmacia LKB Biotechnology Inc.); Sprague-Dawleymale rats (Charles River Breeding Laboratories); N-glycanase (Genzyme);oligo(dT)-cellulose(Collaborative Research);random hexanucleotides,EcoRI dodecamericlinkers, protein
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