Structural and Functional Determinants of Rodent and Human Surfactant Protein A: A Synthesis of Binding and Computational Data

Surfactant protein A (SP-A) provides surfactant stability, first line host defense, and lung homeostasis by binding surfactant phospholipids, pathogens, alveolar macrophages (AMs), and epithelial cells. Non-primates express one SP-A protein whereas humans express two: SP-A1 and SP-A2 with core intra- and inter-species differences in the collagen-like domain. Here, we used macrophages and solid phase binding assays to discern structural correlates of rat (r) and human (h) SP-A function. Binding assays using recombinant rSP-A expressed in insect cells showed that lack of proline hydroxylation, truncations of amino-terminal oligomerization domains, and site-directed serine (S) or alanine (A) mutagenesis of cysteine 6 (C6S), glutamate 195 (E195A), and glutamate 171 (E171A) in the carbohydrate recognition domain (CRD) all impaired SP-A binding. Replacement of arginine 197 with alanine found in hSP-A (R197A), however, restored the binding of hydroxyproline-deficient rSP-A to the SP-A receptor SP-R210 similar to native rat and human SP-A. In silico calculation of Ca++ coordination bond length and solvent accessibility surface area revealed that the “humanized” R197A substitution alters topology and solvent accessibility of the Ca++ coordination residues of the CRD domain. Binding assays in mouse AMs that were exposed to either endogenous SP-A or hSP-A1 (6A2) and hSP-A2 (1A0) isoforms in vivo revealed that mouse SP-A is a functional hybrid of hSP-A1 and hSP-A2 in regulating SP-A receptor occupancy and binding affinity. Binding assays using neonatal and adult human AMs indicates that the interaction of SP-A1 and SP-A2 with AMs is developmentally regulated. Furthermore, our data indicate that the auxiliary ion coordination loop encompassing the conserved E171 residue may comprise a conserved site of interaction with macrophages, and SP-R210 specifically, that merits further investigation to discern conserved and divergent SP-A functions between species. In summary, our findings support the notion that complex structural adaptation of SP-A regulate conserved and species specific AM functions in vertebrates

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency–Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research

SP-R210 (Myo18A) Isoforms as Intrinsic Modulators of Macrophage Priming and Activation

<div><p>The surfactant protein (SP-A) receptor SP-R210 has been shown to increase phagocytosis of SP-A-bound pathogens and to modulate cytokine secretion by immune cells. SP-A plays an important role in pulmonary immunity by enhancing opsonization and clearance of pathogens and by modulating macrophage inflammatory responses. Alternative splicing of the <i>Myo18A </i>gene results in two isoforms: SP-R210_S and SP-R210_L, with the latter predominantly expressed in alveolar macrophages. In this study we show that SP-A is required for optimal expression of SP-R210_L on alveolar macrophages. Interestingly, pre-treatment with SP-A prepared by different methods either enhances or suppresses responsiveness to LPS, possibly due to differential co-isolation of SP-B or other proteins. We also report that dominant negative disruption of SP-R210_L augments expression of receptors including SR-A, CD14, and CD36, and enhances macrophages’ inflammatory response to TLR stimulation. Finally, because SP-A is known to modulate CD14, we used a variety of techniques to investigate how SP-R210 mediates the effect of SP-A on CD14. These studies revealed a novel physical association between SP-R210_S, CD14, and SR-A leading to an enhanced response to LPS, and found that SP-R210_L and SP-R210_S regulate internalization of CD14 via distinct macropinocytosis-like mechanisms. Together, our findings support a model in which SP-R210 isoforms differentially regulate trafficking, expression, and activation of innate immune receptors on macrophages.</p></div

Depletion of SP-R210_L differentially enhances expression of innate immune receptors in macrophages.

<p>Control and SP-R210_L(DN) cells were analyzed by flow cytometry using indicated APC (A) or PE-conjugated antibodies (B, C) (n = 4–8). (D) mRNA levels of indicated receptors in SP-R210_L(DN) cells relative to control cells were determined by qRT-PCR (n = 4 independent experiments performed in duplicate, **p<0.02, ***p<0.005).</p

Proposed interaction of SP-R210 isoforms with CD14 and SR-A in macrophage activation.

<p>SP-R210_L mediates macropinocytosis of LPS-CD14 through interaction with Rac1 resulting in endosomal LPS delivery to TLR-4 and downstream activation of NFκB. Subsequent degradation of LPS results in decrease NFκB signaling. SP-R210_L-mediated macropinocytosis and signaling are sensitive to both EIPA and NSC23766. TLR-4 signaling from the cell-surface is sensitive to dynasore. Inhibition of SP-R210_L expression results in formation of the SP-R210_S-CD14-SR-A complex. Binding of LPS results in macropinocytosis-like internalization of the SP-R210_S-CD14-SR-A complex and activation of a feed-forward inflammatory pathway that depends on activation of Rac1 by SR-A. The SP-R210_S-CD14-SR-A pathway is sensitive to NSC23766 but not EIPA.</p

Activation of TLR4 signaling in control and SP-R210_L(DN) cells.

<p>Macrophages were stimulated with 100 ng/mL LPS and harvested at indicated time points. Non-stimulated (NS) and stimulated cells were harvested and processed for Western blot analysis. Blots were probed with IRAK-1 (A) or NFκB p65 (B) antibodies, stripped, and then reprobed with IκB or phosphorylated p65 antibodies, respectively. Blots were stripped and then re-probed with tubulin as loading control. Densitometry analysis compared the levels of phosphorylated p65 relative to tubulin (C). Blots in A and B are representative of 2–4 independent experiments. Data shown in C are means±SD, n = 2- independent experiments. *p<0.05.</p