Lipid-modified Protein Research Articles

Differential posttranslational processing confers intraspecies variation of a major surface lipoprotein and a macrophage-activating lipopeptide of Mycoplasma fermentans.

The malp gene of Mycoplasma fermentans is shown to occur in single copy but to encode two discrete translated forms of lipid-modified surface protein that can be differentially expressed on isolates within this species: MALP-2, a 14-amino-acid (2-kDa) lipopeptide with potent macrophage-stimulatory activity (P. F. Mühlradt, M. Kiess, H. Meyer, R. Süssmuth, and G. Jung, J. Exp. Med. 185:1951-1958, 1997), and MALP-404, an abundant, full-length (404-amino-acid) surface lipoprotein of 41 kDa, previously designated P41 (K. S. Wise, M. F. Kim, P. M. Theiss, and S.-C. Lo, Infect. Immun. 61:3327-3333, 1993). The sequences, transcripts, and translation products of malp were compared between clonal isolates of strains PG18 (known to express P41) and II-29/1 (known to express high levels of MALP-2). Despite conserved malp DNA sequences containing full-length open reading frames and expression of full-length monocistronic transcripts in both isolates, Western blotting using a monoclonal antibody (MAb) to the N-terminal MALP-2 peptide revealed marked differences in the protein products expressed. Whereas PG18 expressed abundant MALP-404 with detectable MALP-2, II-29/1 revealed no MALP-404 even in samples containing a large comparative excess of MALP-2. Colony immunoblots with the MAb showed uniform surface expression of MALP-2 in II-29/1 populations. A second MAb to an epitope of MALP-404 outside the MALP-2 sequence predictably failed to stain II-29/1 colonies but uniformly stained PG18 populations. Collectively, these results provide evidence for novel posttranscriptional (probably posttranslational) processing pathways leading to differential intraspecies expression of a major lipoprotein, and a potent macrophage-activating lipopeptide, on the surface of M. fermentans. In the course of this study, a striking conserved motif (consensus, TD-G--DDKSFNQSAWE--), designated SLA, was identified in MALP-404; this motif is also distributed among selected lipoproteins and species from diverse bacterial genera, including Bacillus, Borrelia, Listeria, Mycoplasma, and Treponema. In addition, malp was shown to flank a chromosomal polymorphism. In eight isolates of M. fermentans examined, malp occurred upstream of an operon encoding the phase-variable P78 ABC transporter; but, in three of these isolates, a newly discovered insertion sequence, IS1630 (of the IS30 class), was located between these genes.

LolA-dependent Release of a Lipid-modified Protein from the Inner Membrane of Escherichia coli Requires Nucleoside Triphosphate

The outer membrane-directed lipoproteins are released from the inner membrane of Escherichia coli as a complex with LolA, a periplasmic chaperone. The LolA-dependent release of lipoproteins is critical for lipoprotein sorting as it depends on the outer membrane-specific sorting signal. To clarify molecular events involved in the LolA-dependent lipoprotein release, we attempted to establish an in vitro assay system. The major outer membrane lipoprotein (Lpp) was found to lose its release competence soon after being processed to mature Lpp in the inner membrane and therefore could not be used as a substrate for an in vitro system. An Lpp derivative, L10P, was constructed and found to retain the release competence long after its maturation. L10P was synthesized and radiolabeled in spheroplasts in the absence of LolA; therefore, it remained anchored to the inner membrane of spheroplasts. Right-side out membrane vesicles containing L10P were then prepared and used to examine the release of L10P. In addition to LolA, L10P release absolutely required nucleoside triphosphate (NTP). A non-hydrolyzable NTP analogue strongly inhibited the NTP-dependent release. The outer membrane-specific sorting signal was essential for the in vitro release of L10P. Furthermore, L10P released in vitro was specifically incorporated into the outer membrane. These results indicate that the in vitro release of L10P represents an in vivo reaction and requires energy.

Antigenic topology of the P29 surface lipoprotein of Mycoplasma fermentans: differential display of epitopes results in high-frequency phase variation

Antibodies to P29, a major lipid-modified surface protein of Mycoplasma fermentans, reveal phase variation of surface epitopes occurring with high frequency in clonal lineages of the organism. This occurs despite continuous expression of the entire epitope-bearing P29 product (detected by Western immunoblotting) and contrasts with phase variation of other surface antigens mediated by differential expression of proteins. To understand the structure and antigenic topology of P29, the single-copy p29 gene from strain PG18 was cloned and sequenced. The gene encodes a prolipoprotein containing a signal sequence predicted to be modified with lipid and cleaved at the N-terminal Cys-1 residue of the mature P29 lipoprotein. The remaining 218-residue hydrophilic sequence of P29 is predicted to be located external to the single plasma membrane. Additional Cys residues at positions 91 and 128 in the mature protein were shown to form a 36-residue disulfide loop by selectively labeling sulfhydryl groups that were liberated only after chemical reduction of monomeric P29. Two nearly identical charged amino acid sequences occurred in P29, within the disulfide loop and upstream of this structure. Two distinct epitopes binding different monoclonal antibodies were associated with opposite ends of the P29 protein, by mapping products expressed in Escherichia coli from PCR-generated 3' deletion mutations of the p29 gene. Each monoclonal antibody detected high-frequency and noncoordinate changes in accessibility of the corresponding epitopes in colony immunoblots of clonal variants, yet sequencing of the p29 gene from these variants and analysis of disulfide bonds revealed no associated changes in the primary sequence or disulfide loop structure of P29. These results suggest that P29 surface epitope variation may involve masking of selected regions of P29, possibly by other surface components undergoing phase variation by differential expression. Differential masking may be an important mechanism for altering the antigenic or functional surface topology of this mycoplasma and other wall-less mycoplasmas.

Identification and mapping of an immunogenic region of Mycoplasma hyopneumoniae p65 surface lipoprotein expressed in Escherichia coli from a cloned genomic fragment

A previously characterized lipid-modified amphiphilic surface protein of Mycoplasma hyopneumoniae, p65, has been defined by its reaction with a surface-binding monoclonal antibody (MAb) and by its exclusive partitioning into the detergent phase during Triton X-114 phase fractionation (K. S. Wise and M. F. Kim, J. Bacteriol. 169:5546-5555, 1987). In the current study, polyclonal mouse antibody (PAb) to gel-purified p65 was used to identify recombinant phage plaques expressing p65-related epitopes. Several characteristic partial tryptic fragments of p65 were recognized by both PAb and p65 and MAb to p65, but the PAb population specifically eluted from recombinant phage plaques bound only epitopes restricted to the largest of these fragments. Graded carboxypeptidase-Y digestion of intact M. hyopneumoniae generated C terminally truncated peptides that were recognized by PAb to p65 and MAb to p65, indicating that the C terminus and much of the adjoining region of p65 were present and accessible on the external face of the membrane. However, antibody eluted from recombinant phage plaques bound only to the largest truncated polypeptide, suggesting that a recombinant product corresponding to the C-terminal region of p65 was expressed in Escherichia coli. A 19-kilodalton recombinant protein (p19), which was recognized by PAb to p65 but not by MAb to p65, was detected in recombinant phage lysates. Serum antibodies from swine taken after, but not before, experimentally induced M. hyopneumoniae pneumonia preferentially recognized the native, amphiphilic p65 lipoprotein and also bound specifically to the p19 recombinant product. This confirmed that the p65 lipoprotein is a major immunogen of M. hyopneumoniae recognized during disease and identified its C-terminal region as an immunogenic domain.

An amphitropic cAMP-binding protein in yeast mitochondria. 2. Phospholipid nature of the membrane anchor.

We describe the first example of a mitochondrial protein with a covalently attached phosphatidylinositol moiety acting as a membrane anchor. The protein can be metabolically labeled with both stearic acid and inositol. The stearic acid label is removed by phospholipase D whereupon the protein with the retained inositol label is released from the membrane. This protein is a cAMP receptor of the yeast Saccharomyces cerevisiae and tightly associated with the inner mitochondrial membrane. However, it is converted into a soluble form during incubation of isolated mitochondria with Ca2+ and phospholipid (or lipid derivatives). This transition requires the action of a proteinaceous, N-ethylmaleimide-sensitive component of the intermembrane space and is accompanied by a decrease in the lipophilicity of the cAMP receptor. We propose that the component of the intermembrane space triggers the amphitropic behavior of the mitochondrial lipid-modified cAMP-binding protein through a phospholipase activity.