The Murein-Lipoprotein Linkage in the Cell Wall of Escherichia coli

Abstract

After digestion of the murein-lipoprotein complex (rigid layer) of lysozyme the solubilized lipoprotein contained 2 disaccharide units of the murein per 1 lipoprotein molecule. From purely enzymatic degradation of the murein-lipoprotein complex with pronase and lysozyme the peptide GlcNAc-MurNAc-L-Ala-D-Glu-Dpm-Lys-Arg was isolated, where MurNAc = N-acetyl-muramic acid and Dpm = meso-2,6-diaminopimelic acid. The missing D-alanine in this repeating unit of the murein is replaced by the lysyl-arginine dipeptide of the lipoprotein. The lipoprotein molecules are therefore linked to the carboxyl group of the optical L-center of the diaminopimelic acid of the murein. . Therefore by the use of both enzymatic procedures one obtains one of the two macromolecules (the murein or the lipoprotein) undegraded, to which a small part of the other remains covalently bound. Previously we isolated from partial acid hydrolysates of murein-lysyl-arginine degradation products with which we could show that the lipoprotein molecules are bound to the diaminopimelic acid of nearly every tenth disaccharide unit of the murein (Dpm-Lys-Arg). It was also found that none of the degradation products bearing lysine and arginine contained D-alanine but this could have been lost by the partial acid hydrolysis. By a purely enzymatic procedure we shall show here that the disaccharide units of the murein which bear the lipoprotein molecules in fact are devoid of D-alanine and that the lipoprotein molecules actually replace the D-alanine a t the carboxyl group of the optical L-center of diaminopimelic acid. I n addition we isolated after lysozyme digestions of the mureinlipoprotein complex the lipoprotein in a soluble form and it contained two disaccharide units of the murein. MATERIALS AND METHODS Enzymatic DegradationsLysozyrne Digestion. 50 mg of the murein-lipoprotein complex prepared as described [4,7] were suspended in 100 ml 0.01 M Tris-C1 pH 7.4 and digested with 1 mg of lysozyme (Boehringer Mannheim GmbH, Mannheim) for 15 h at 34". The solution was centrifuged for 30 min a t 37 000 x g, the supernatant was either acidified to pH 3.7 or concentrated to 2 ml on a rotary evaporator for column chromatography on Sephadex G-75. The precipitate of the acidified solution was spun down, washed twice with 0.1 M acetic acid and lyophilized.Promse-Lysozyme Digestion. Incubation of the murein-lipoprotein complex with pronase P (Serva, Heidelberg) followed the procedure described [7]. After washing the resulting murein-lysyl-arginine with 4OI0 sodium dodecyIsulfate and then 4 times with water, the digestion with lysozyme followed the procedure described above. The solution of the degradation products was concentrated to about 15m1, acidified to pH 2 and run on a column of Dowex 50x2. The collected peak fractions, identified by ninhydrin reaction after alkaline hydrolysis, were tested for homogeneity by paper chromatography in the system butanol-acetic acid-water (4: 1 : 5 , upper phase) and aliquots were run on the amino acid analyzer as described [4,7]....

Sequence of the Murein · Lipoprotein and the Attachment Site of the Lipid

“…After a single step of Edman degradation arginine was lost as previously found [3,4] indicating arginine a t the N-terminal end of peptides 56 and 57. After Edman degradation without hydrolysis free lysine was released from peptide 56 but not from peptide 57.…”

“…The previously found release of arginine upon incubation with carboxypeptidase B [3,4] was again confirmed several times, e.g. after a 4.5-h splitting 64O/, free arginine was obtained from peptide 57 and a similar amount from peptide 56.…”

“…The lipoprotein is always analysed with 1-2 murein repeating units still attached to it [3,4] and correspondingly only 1-2 glutamate residues were subtracted from the lipoprotein analysis. From a partial acid hydrolysate of such a lipoprotein we isolated a compound with the composition glucosamine-glutamate 1 : I.…”

Antigenic Determinants of Murein Lipoprotein and Its Exposure at the Surface of Enterobacteriaceae

“…To determine the antigenic sites of this lipoprotein several tests were performed. We first incubated anti-lipoprotein antiserum with pure muropeptides, prepared as described previously [12]. The lipoprotein titer of an antiserum was only slightly reduced (about one hemagglutinin unit) when 1 pg of muropeptides was added for serum absorption ; higher concentrations (up to 100 pg muropeptides) led only to a slight further titer decrease.…”

“…The preparation of the various lipoprotein samples has been described in detail elsewhere : lipoprotein I (containing two or three muropeptides) is obtained by degradation of murein with lysozyme [12]; lipoprotein I1 (i.e. free of muropeptides and lacking the C-terminal peptide Tyr-Arg-Lys) is released from murein by treatment with trypsin [13]; isolation of lipoprotein IV (i.e.…”