Recent studies provided evidence that in AM from C57Bl/6 mice, MARCO plays a predominant role in binding of toxic CSiO2
particles (Hamilton et al., 2006
). The ability of MARCO to bind inert nontoxic TiO2
particles was first reported by Kobzik and coworkers (Palecanda et al., 1999
). These observations raise an important question as to why, despite binding to a common receptor MARCO, certain inorganic particles such as CSiO2
are toxic to the AM while TiO2
particles are not (). We hypothesized that the differences in the apoptotic outcome in response to these inorganic particles may, at least in part, be related to differences in binding of these particles to MARCO. To understand the differences in binding of environmental particles to MARCO, the purpose of this study was to define the particle-binding domain of MARCO and map some of the determinants for individual particle binding to MARCO.
To explore the possibility that crystalline and amorphous forms of silica, as well as TiO2, bind to distinct motifs in the receptor MARCO, a transfected cell line model was developed. For these studies, CHO cells expressing full-length MARCO or various MARCO mutants were used. The cell-surface expression of the full-length MARCO and mutants was confirmed by cell-surface biotinylation (). The results of the binding studies conducted with full-length MARCO-transfected cells showed that all three particles bound to MARCO (). The fact that the MARCO-specific antibody, which binds to an epitope in the SRCR domain, significantly inhibited the binding of all three particles to MARCO suggested that the SRCR domain was the particle-binding domain of MARCO. This finding was confirmed by the observation that the MARCO mutant without the SRCR domain failed to bind any of the particles (). Consequently, the data established that all these particles require the SRCR domain for binding. Apoptosis assays with transfected CHO cells further showed that the CSiO2 binding to the SRCR domain of MARCO was required for its cytotoxicity (), whereas treatment with ASiO2 and TiO2 did not induce apoptosis in the full-length MARCO-transfected cells (data not shown) despite efficient binding (). The results support the hypothesis that the SRCR domain is the binding domain for environmental particles.
The next question was whether the RGR motif within the SRCR domain would be sufficient for particle binding. The M442 mutant containing the RGR motif showed significant ability (distinctly reduced compared to full-length MARCO) to bind only the ASiO2
particles (). The CSiO2
particles did not bind to the M442 mutant−expressing cells. The RGR motif within the SRCR domain has been previously shown to be sufficient for bacterial binding (Brannstrom et al., 2002
). Importantly, this finding suggests that ASiO2
is unique with respect to the RGR motif in contrast to CSiO2
and, hence, binds distinctly to MARCO.
Competitive binding studies were conducted to further investigate how each particle binds to the SRCR domain of MARCO. For these studies, cells were pretreated with CSiO2 or TiO2 particles prior to incubation with ASiO2 particles. Considering relatively large sizes of the particles, the complete inhibition of ASiO2 binding by CSiO2 was not unexpected (). Furthermore, this observation does not negate the proposed role of the RGR motif as being sufficient for ASiO2 binding. It should be kept in mind while interpreting these results that all these particles are very large with respect to MARCO. Therefore, the observation that TiO2 did not completely block ASiO2 binding is more difficult to explain (). Nevertheless, it indicates a divergence in the requirements between both the silica particles and TiO2 in binding to MARCO. This is not a classic case of ligand receptor binding but may require multiple MARCO receptors interacting with these rather large ligands.
Divalent cation-binding properties of certain receptors such as low density lipoprotein receptors are often exploited in nature to regulate complex biological events such as receptor-ligand interaction, endocytosis, and dissociation of the ligand from the receptor (Dirlam-Schatz and Attie, 1998
). Recently, the SRCR domain of MARCO was shown to contain an acidic and a distinct basic cluster of amino acids, both the clusters were reported to be important for ligand binding. The crystallized SRCR domain of MARCO contained a divalent cation in the acidic cluster (Ojala et al., 2007
). In the current study, divalent cations (Ca+2
) were necessary only for TiO2
binding (), whereas the other two inorganic particles (CSiO2
) did not depend on the presence of divalent cations (). Calcium binding to the cysteine-rich domain of a particular protein has been shown to stabilize the protein conformation (Handford et al., 1990
; Knott et al., 1996
; Thielens et al., 1988
). Bound calcium might cause a conformational change in the SRCR domain and expose certain amino acid residues leading to more efficient binding. The data suggest that either the TiO2
binds to the acidic cluster (containing the divalent cations) of the SRCR domain or the divalent cation binding to the SRCR domain leads to a distinct conformational changes in the binding domain facilitating TiO2
binding. It should be noted that changes in dispersion medium such as divalent cations will affect the zeta potential of all three particles. However, addition of divalent cations would affect all particles in a relatively similar manner. Therefore, it is most likely that the divalent cations in this study act on the SRCR domain of MARCO. Taken together, the results (, , and ) emphasize that the three different particles in the study show significant differences in binding to MARCO.
The differences in the pathological outcomes after exposure to each particle are speculated to be related to the differences in the physical properties of the particles. (Johnston et al., 2000
; Thakur et al., 2008
). Therefore, the particles were characterized and analyzed for their size, shape, and surface charge (). The analysis suggested that there were no major differences in size of the particles. The overall surface charge or the zeta potential of the silica particles was essentially identical (−) 16.2 to (−) 17.8 mV. The TiO2
particles were the most negative particles with zeta potential (−) 47.9. While the net surface charge of the particles could be measured by zeta potential measurements, the surface charge distribution (order) could not be determined. While it appears that TiO2
differs from the two silica particles in how it binds to MARCO and, therefore, could explain the difference in toxicity between TiO2
, the difference between ASiO2
appears to be subtle. They are similar in relative size and surface charge but differ in shape and crystal structure (). It is possible that one or both the properties contribute to the difference in toxicity between the two silica particles. The only difference in binding appeared to be the sufficiency of the RGR motif for ASiO2
binding. The assumption made in these studies is that the two silica particles cause difference in conformational change in MARCO such that CSiO2
causes apoptosis while ASiO2
does not. As stated above, the difference in shape and crystal structure could affect silica binding to MARCO causing differences in conformation and downstream signaling. Without more data, the importance of shape or crystal structure is speculative. As yet the signaling pathways initiated by receptor MARCO are not completely elucidated, which makes the aforementioned theory difficult to test experimentally.
We propose that factors such as presence of divalent cations, shape of the particle, and crystal structure (the distribution or the order of surface charge) on the particle may play an important role in determining toxic or nontoxic binding of particles. Studies conducted with more diverse sizes and surface charge, of one particle could further strengthen these conclusions. Nevertheless, the results obtained in the current study provide strong support to the notion that environmental particles bind to distinct motifs in the SRCR domain of MARCO, which is influenced by individual physical properties. The implications of these observations are that each particle-MARCO interaction may trigger unique conformational changes in the receptor, which might influence the recruitment of different intracellular proteins leading to diverse biological responses.