The amino acid sequence of Neisseria lactamica PorB surface-exposed loops influences Toll-like receptor 2-dependent cell activation.

Toll-like receptors (TLRs) play a major role in host mucosal and systemic defense mechanisms by recognizing a diverse array of conserved pathogen-associated molecular patterns (PAMPs). TLR2, with TLR1 and TLR6, recognizes structurally diverse bacterial products such as lipidated factors (lipoproteins and peptidoglycans) and nonlipidated proteins, i.e., bacterial porins. PorB is a pan-neisserial porin expressed regardless of organisms' pathogenicity. However, commensal Neisseria lactamica organisms and purified N. lactamica PorB (published elsewhere as Nlac PorB) induce TLR2-dependent proinflammatory responses of lower magnitude than N. meningitidis organisms and N. meningitidis PorB (published elsewhere as Nme PorB). Both PorB types bind to TLR2 in vitro but with different apparent specificities. The structural and molecular details of PorB-TLR2 interaction are only beginning to be unraveled and may be due to electrostatic attraction. PorB molecules have significant strain-specific sequence variability within surface-exposed regions (loops) putatively involved in TLR2 interaction. By constructing chimeric recombinant PorB loop mutants in which surface-exposed loop residues have been switched between N. lactamica PorB and N. meningitidis PorB, we identified residues in loop 5 and loop 7 that influence TLR2-dependent cell activation using HEK cells and BEAS-2B cells. These loops are not uniquely responsible for PorB interaction with TLR2, but NF-κB and MAP kinases signaling downstream of TLR2 recognition are likely influenced by a hypothetical "TLR2-binding signature" within the sequence of PorB surface-exposed loops. Consistent with the effect of purified PorB in vitro, a chimeric N. meningitidis strain expressing N. lactamica PorB induces lower levels of interleukin 8 (IL-8) secretion than wild-type N. meningitidis, suggesting a role for PorB in induction of host cell activation by whole bacteria.

Limited proteolysis and biophysical characterization of the lipovitellin homology region in apolipoprotein B.

Apolipoprotein B (apoB) is the essential nonexchangeable protein in chylomicrons and very low-density lipoprotein-derived lipoprotein particles, including low-density lipoprotein (LDL). ApoB has been a key target for cardiovascular research because of its essential role in the assembly, secretion, delivery, and receptor binding of LDL. The three-dimensional structure of apoB has not been determined. However, the N-terminal region of apoB is homologous to the lipid storage protein lipovitellin, which allows the modeling of this region based on the X-ray structure of lipovitellin. The model of the N-terminal 17% of apoB (B17) suggests that, like lipovitellin, B17 consists of an N-terminal beta-barrel domain, a helical domain, and a beta-sheet domain (C-sheet). Here we test the validity of this model by limited proteolysis of B17 and the characterization of individual domains expressed in Escherichia coli and insect cell systems that are consistent with the model and proteolysis data. Circular dichroism studies of the individual domains indicate that they are folded and their secondary structures are in agreement with the model. We find that the helical domain and C-sheet of apoB interact with each other in vitro, suggesting a strong interaction between these two domains, even without a covalent peptide bond linkage. Our data suggest that the three lipovitellin-like domains exist in B17. Furthermore, the domains fold independently with secondary structures and stabilities like those of intact B17.