The primary structure of a covalent lipoglycoprotein (murein . lipoprotein) from the cell wall of Escherichia coli is presented. The amino acid sequence consists of57 residues. The lipid is attached to the N-terminal serine and the murein is bound to the 6-amino group of the C-terminal lysine of the lipoprotein. The repetitive design of the sequence suggcsts a very conservative evolution in which a structural gene coding for 15 amino acids was duplicated once and then only half of this gene, thus coding for 7 amino acids was fused four times with the first 29 amino acids. Some deletions but only few exchanges of amino acids apparently occurred.This structural membrane lipoprotein aggregates strongly when released from the murein by lysozyme digestion. The molecular weight was therefore determined in 2O/,, 0.5O/, and O.lo/o dodecylsulfate by polyacrylamide gel electrophoresis and gel chromatography. The molecular weight of 13000 thus found deviated from that of I0000 calculated from the sum of the amino acids, the lipid as known so far and the part of the murein remaining bound to the lipoprotein. This was not due to a different amount of dodecylsulfate binding by the lipoprotein compared with proteins containing no lipid but may be due to a more spherical shape of the small molecule in contrast to the rod-like shape of standard proteins in dodecylsulfate.The lipoprotein which is covalently attached to the murein [I] of the outer membrane of Escherichia coli [2--51 is one of thc few membrane proteins isolated so far in pure form. Even soluble lipoproteins, as they occur e.g. in the serum, are difficult to purify and only rather limited information about their structure is available today [6,7]. In addition, in the lipoprotein we are working, the lipid is very probably covalently bound [2,8] which was not found in any other lipoprotein mixture studied so far. I n order to elucidate the attachment site of the lipid and the mode of binding on the murein, we studied the amino acid sequence of the polypeptide chain. In a previous paper [Y] we discussed some features of the unusual and very interesting results especially with regard to the repetitive design of the sequence and the relevance of this study to the structure of membrane proteins and membranes in general. Now we present the experimental evidence together with a full account of the results.
The amino-acid sequence of the murein-lipoprotein of the Escherichia coli cell wall is presented. This protein is covalently bound to a lipid component as well as to the murein (peptidoglycan, mucopeptide). The sequence is also highly repetitive. At the N-terminal portion, there are three adjacent almost identical sequences, indicating repeated duplication of a gene coding originally for 15 amino acids. The C-terminal part of the polypeptide chain is more variable but still shows striking homology when certain sequence gaps are introduced. The lipid is bound to the N-terminal serine of the dipeptide (Ser-Ser) that extends from the repetitive sequence. At the C-terminal end where the murein is bound, a tripeptide extends from the repetitive portion. Here there are several basic amino acids and the only aromatic amino acid in the lipoprotein. The sequence is Lys-Tyr-Arg-Lys. The linkage to the murein is formed between the ε-amino group of the C-terminal lysine and the carboxyl group of the optical L-center of meso-diaminopimelic acid. The polypeptide chain is composed of 57 amino acids and lacks glycine, proline, cysteine, phenylalanine, histidine, and tryptophan. 63% of the amino acids are hydrophilic, but because of the covalently linked lipid this structural membrane protein has very hydrophobic properties.
Murein lipoprotein from the outer membrane of Escherichia coli could be fixed to erythrocytes without pretreatment of the erythrocytes. Passive hemagglutination or immune hemolysis could thus be used as sensitive assays to determine antibodies against lipoprotein. In rabbit antisera prepared against whole cells of E. coli, Sulmonellu, Arizona and Shigellu antibodies against lipoprotein were present. The respective titers were lowest in encapsulated smooth strains and highest in rough mutants. Antisera against deep rough mutants showed even higher anti-lipoprotein titers than anti-R-lipopolysaccharide titers. Correspondingly, absorption of lipoprotein antibodies with enterobacterial strains was most pronounced with deep rough mutants and lowest with smooth strains. Lipoprotein becomes increasingly an immunogen as well as an antigen the more sugar residues are missing in the lipopolysaccharide on the cell surface. In wild-type cells lipoprotein is buried in the outer membrane; its exposure in mutant cells is related to defects at the cell surface.Antisera were prepared in rabbits against erythrocytes coated with various lipoprotein samples which differ at the C-terminal end of the polypeptide chain. Antibodies against lipoprotein containing two attached muropeptides (called lipoprotein I) are directed against the muropeptide attachment region. Antibodies against lipoprotein ending with lysine-55 (lipoprotein 11) were directed specifically against this terminal lysine residue. No serological reaction was observable after splitting off this lysine residue by carboxypeptidase B. Remarkably this antiserum against lipoprotein I1 neither reacted with the free lipoprotein ending with lysine-58 (lipoprotein 1V) nor with the muropeptide containing lipoprotein I. Antisera against the lipoprotein lacking lysine-55 (lipoprotein 111) reacted with all lipoprotein samples as did the antibodies in antisera prepared against whole heat-killed cells, thus indicating a common binding site located in the polypeptide chain towards the N-terminal end. No indication of an antigenic reactivity of the covalently linked lipoid was obtained.sodium dodecylsulfate and acetone reacted strongly with antisera against the native lipoprotein. The recovery of the antigenic sites demonstrates the remarkable stability of the native conformation.The lipoprotein is a new common antigen for closely related Enterobacteriaceae. Lipoprotein antisera might be useful for characterization of outer membrane mutants of Enterobacreriaceue.Lipoprotein treated with boiling 4
Abo~xl 2 X 105 .lipoprotein. n.a,~lecules] , 1]] a.le covalenvly b~unfl to the mmein t2] (peplidoglycan) of ~e .outer membzane ofg. eoli and zela~t.ed en~erobac-~,ea-iaceae :I3, 4]. Twice ,this am,oum of lipop~otem was found free in I.tle eel] eJav~l.ope [5] without specification w'hethez d.xe f~ee-fo~"m occurs ~n ,th.e o~a,Iex membrane, the cy~.op]a~mic naelnbzmae ol 5n bo~h. We found by immunol0giea3 means tha~t rnor:e ,lhan . 90% of the mta~ fipopmlein is localized in the omei membrane I6]. The ]ipoproI.eiu naomi plobaMy is syn~.e~ized in the cytoplasm or khe cytoplasmic side of lhe cytoplasmi,c membrane and the :questi13n arises, how i,I is Irmasfelred thlough ~he cytoplasmic rnenv blan.e into the .ouler membrane an d how it is built into ~h,e rnu~ein? The two s.~mpl'es~ .al.te~nativ.e~ a~e: a mu~ein-lepeat-;ng unit is already fixed to the lip~-p:rotem in the ,cytoplasmic memb.~a.ne am,d inco~r,p13-la'ted as such into the murebn ox ,the lip.aplo~ein is "trausf~r~e:d to the preformed rnm'ein in the omei lnemblane. In ~irr.o studies with soluble and par, tima-]a~_e ,enzy__me systems ievealed the pathway-of naurein :syn,thesi's up ,t.o the s,tage whe,i.e ~h,e repeating uni~, a disa¢charide-pen,apep'fide, is bound ~o a lipid ,c~axri.ei, a 'C55-potyisup~en.oid alcohol, by a py~,oph.osphate linkage. Polymeli~atti.on of I2b.e/dpid-link,ed ~epea~ing unit to peplidogly,can .chains and cross--":.inkage between pe:pfid0glyean chains, ~hich is inhibited by penicillin, was :also achieved in ~itro .(see review [7] ).:Ou~/n ~ivo atudies described here we;e eoncemed with the questi:onswlaeth¢~ ]ipolaZOXein ~Salready tin.k.ed to ~ murein-iepeating urtit in .the cy.t.oplasmic 'membrane.,:whether :muz.c~peplides ark polymelizedo~ ,cr,oss-~ked :in ',the ,cytoI,dasmie ,.memtH.ane and ~,then tiansfen~ed as ,0]]gorn,e~ to ~,e ou~,er membrane ,and wheth~i ]ipaprotein is incorporated tandomly ,o~ at ~pecific sites into the mu~in of .~the our,m" rnemb~ane. Melhods~. cotiW7 (dap-, ]
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