A Genetic Engineering Solution to the ��Arginine Conversion Problem�� in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Stable isotope labeling by amino acids in cell culture (SILAC) provides a straightforward tool for quantitation in proteomics. However, one problem associated with SILAC is the in vivo conversion of labeled arginine to other amino acids, typically proline. We found that arginine conversion in the fission yeast Schizosaccharomyces pombe occurred at extremely high levels, such that labeling cells with heavy arginine led to undesired incorporation of label into essentially all of the proline pool as well as a substantial portion of glutamate, glutamine, and lysine pools. We found that this can be prevented by deleting genes involved in arginine catabolism using methods that are highly robust yet simple to implement. Deletion of both fission yeast arginase genes or of the single ornithine transaminase gene, together with a small modification to growth medium that improves arginine uptake in mutant strains, was sufficient to abolish essentially all arginine conversion. We demonstrated the usefulness of our approach in a large scale quantitative analysis of proteins before and after cell division; both up- and down-regulated proteins, including a novel protein involved in septation, were successfully identified. This strategy for addressing the “arginine conversion problem” may be more broadly applicable to organisms amenable to genetic manipulation.Stable isotope labeling by amino acids in cell culture (SILAC)¹ (1) is one of the key methods for large scale quantitative proteomics (2, 3). In SILAC experiments, proteins are metabolically labeled by culturing cells in media containing either normal (“light”) or heavy isotope-labeled amino acids, typically lysine and arginine. Peptides derived from the light and heavy cells are thus distinguishable by mass spectrometry and can be mixed for accurate quantitation. SILAC is also possible at the whole-organism level (4).An inherent problem in SILAC is the metabolic conversion of labeled arginine to other amino acids, as this complicates quantitative analysis of peptides containing these amino acids. Arginine conversion to proline is well described in mammalian cells, although the extent of conversion varies among cell types (5). When conversion is observed, typically 10–25% of the total proline pool is found to contain label (6–11). Arginine conversion has also been reported in SILAC experiments with budding yeast Saccharomyces cerevisiae (3, 12, 13).Because more than 50% of tryptic peptides in large data sets contain proline (7), it is not practical simply to disregard proline-containing peptides during quantitation. Several methods have been proposed to either reduce arginine conversion or correct for its effects on quantitation. In some cell types, arginine conversion can be prevented by lowering the concentration of exogenous arginine (6, 14–16) or by adding exogenous proline (9). However, these methods can involve significant changes to growth media and may need to be tested for each experimental condition used. Given the importance of arginine in many metabolic pathways, careful empirical titration of exogenous arginine concentration is required to minimize negative effects on cell growth (14). In addition, low arginine medium can lead to incomplete arginine labeling, although the reasons for this are not entirely clear (7). An alternative strategy is to omit labeled arginine altogether (3, 13, 17), but this reduces the number of quantifiable peptides. Correction methods include using two different forms of labeled arginine (7) or computationally compensating for proline-containing peptides (11, 12, 18). Ultimately, none of these methods address the problem at its root, the utilization of arginine in cellular metabolism.To develop a differential proteomics work flow for the fission yeast Schizosaccharomyces pombe, we sought to adapt SILAC for use in this organism, a widely used model eukaryote with excellent classical and reverse genetics. Here we describe extremely high conversion of labeled arginine to other amino acids in fission yeast as well as a novel general solution to the problem that should be applicable to other organisms. As proof of principle, we quantitated changes in protein levels before and after cell division on a proteome-wide scale. We identified both up- and down-regulated proteins, including a novel protein involved in septation.     

Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

Serine proteinases in insect plasma have been implicated in two types of immune responses; that is, activation of prophenoloxidase (proPO) and activation of cytokine-like proteins. We have identified more than 20 serine proteinases in hemolymph of the tobacco hornworm, Manduca sexta, but functions are known for only a few of them. We report here functions of two additional M. sexta proteinases, hemolymph proteinases 6 and 8 (HP6 and HP8). HP6 and HP8 are each composed of an amino-terminal clip domain and a carboxyl-terminal proteinase domain. HP6 is an apparent ortholog of Drosophila Persephone, whereas HP8 is most similar to Drosophila and Tenebrio spätzle-activating enzymes, all of which activate the Toll pathway. proHP6 and proHP8 are expressed constitutively in fat body and hemocytes and secreted into plasma, where they are activated by proteolytic cleavage in response to infection. To investigate activation and biological activity of HP6 and HP8, we purified recombinant proHP8, proHP6, and mutants of proHP6 in which the catalytic serine was replaced with alanine, and/or the activation site was changed to permit activation by bovine factor Xa. HP6 was found to activate proPO-activating proteinase (proPAP1) in vitro and induce proPO activation in plasma. HP6 was also determined to activate proHP8. Active HP6 or HP8 injected into larvae induced expression of antimicrobial peptides and proteins, including attacin, cecropin, gloverin, moricin, and lysozyme. Our results suggest that proHP6 becomes activated in response to microbial infection and participates in two immune pathways; activation of PAP1, which leads to proPO activation and melanin synthesis, and activation of HP8, which stimulates a Toll-like pathway.Innate immune systems of mammals and arthropods include extracellular serine proteinase cascade pathways, which rapidly amplify responses to infection and stimulate killing of pathogens. These proteinase-driven processes include the complement system of vertebrates (1, 2) and pathways in arthropods involving proteinases containing amino-terminal clip domains (3). Clip domain proteinases function in blood coagulation (4, 5), activation of prophenoloxidase (proPO) that leads to melanin synthesis (6–9), and stimulation of the Toll pathway to promote synthesis of antimicrobial peptides/proteins (AMPs)² secreted into the hemolymph (10, 11).The serine proteinase systems best characterized in arthropods are the horseshoe crab hemolymph coagulation pathway and the cascade leading to activation of the Toll pathway in dorsal-ventral development in Drosophila (12–14). Recent research also has led to better characterization of the proPO activation pathway in Manduca sexta (7, 15, 16) and the Toll-signaling pathway in the Drosophila immune response (17, 18) and to both the proPO and Toll pathways in the beetle Tenebrio molitor (11, 19).In the proPO activation pathway, soluble pattern recognition proteins initially recognize pathogen-associated molecular patterns such as bacterial peptidoglycan or fungal β-1,3-glucan (20–22). This interaction stimulates the sequential activation of a series of serine proteinases in hemolymph, leading to the activation of proPO-activating proteinase (PAP), also known as proPO activating enzyme (7, 23). Activated PAP converts inactive proPO to PO. PO catalyzes the hydroxylation of monophenols to o-diphenols and the oxidation of o-diphenols to quinones that are involved in microbial killing, melanin synthesis, sequestration of parasites or pathogens, and wound healing (24, 25). Other proteins required for proPO activation are clip-domain serine proteinase homologs (SPHs), whose catalytic serine is replaced with glycine and, therefore, lack proteolytic activity (26, 27). Serine proteinase inhibitors, including members of the serpin superfamily, regulate the activation of proPO by inhibiting the activating proteinases (28, 29).Drosophila clip-domain serine proteinases Persephone, Grass, Spirit, and spätzle-processing enzyme (SPE) participate in the activation of Toll pathway, stimulating synthesis of antimicrobial peptides as an innate immune response (18, 30–32). Although genetic evidence indicates that Persephone and Spirit are upstream of SPE in the cascade, the substrate(s) of Persephone and Spirit have not been identified, and which proteinase directly activates SPE is unknown. Neither is it clear whether these enzymes may be related to the melanization pathway, which involves clip-domain proteinases MP2 and MP1 (33).Here we report the functional characterization of M. sexta HP6 and HP8, probable orthologs of Drosophila Persephone and SPE, respectively. We developed methods to activate purified recombinant proHP6 and proHP8 and discovered that HP6 participates in proPO activation by activating proPAP1 and that both HP6 and HP8 function in a pathway that stimulates the synthesis of AMPs in M. sexta.