Pneumococcal Neuraminidase Substrates Identified through Comparative Proteomics Enabled by Chemoselective Labeling

Neuraminidases (sialidases) are enzymes that hydrolytically remove sialic acid from sialylated proteins and lipids. Neuraminidases are encoded by a range of human pathogens, including bacteria, viruses, fungi, and protozoa. Many pathogen neuraminidases are virulence factors, indicating that desialylation of host glycoconjugates can be a critical step in infection. Specifically, desialylation of host cell surface glycoproteins can enable these molecules to function as pathogen receptors or can alter signaling through the plasma membrane. Despite these critical effects, no unbiased approaches exist to identify glycoprotein substrates of neuraminidases. Here, we combine previously reported glycoproteomics methods with quantitative proteomics analysis to identify glycoproteins whose sialylation changes in response to neuraminidase treatment. The two glycoproteomics methodsperiodate oxidation and aniline-catalyzed oxime ligation (PAL) and galactose oxidase and aniline-catalyzed oxime ligation (GAL)rely on chemoselective labeling of sialylated and nonsialylated glycoproteins, respectively. We demonstrated the utility of the combined approaches by identifying substrates of two pneumococcal neuraminidases in a human cell line that models the blood-brain barrier. The methods deliver complementary lists of neuraminidase substrates, with GAL identifying a larger number of substrates than PAL (77 versus 17). Putative neuraminidase substrates were confirmed by other methods, establishing the validity of the approach. Among the identified substrates were host glycoproteins known to function in bacteria adherence and infection. Functional assays suggest that multiple desialylated cell surface glycoproteins may act together as pneumococcus receptors. Overall, this method will provide a powerful approach to identify glycoproteins that are desialylated by both purified neuraminidases and intact pathogens

Sialidase Specificity Determined by Chemoselective Modification of Complex Sialylated Glycans

Sialidases hydrolytically remove sialic acids from sialylated glycoproteins and glycolipids. Sialidases are widely distributed in nature and sialidase-mediated desialylation is implicated in normal and pathological processes. However, mechanisms by which sialidases exert their biological effects remain obscure, in part because sialidase substrate preferences are poorly defined. Here we report the design and implementation of a sialidase substrate specificity assay based on chemoselective labeling of sialosides. We show that this assay identifies components of glycosylated substrates that contribute to sialidase specificity. We demonstrate that specificity of sialidases can depend on structure of the underlying glycan, a characteristic difficult to discern using typical sialidase assays. Moreover, we discovered that <i>Streptococcus pneumoniae</i> sialidase NanC strongly prefers sialosides containing the Neu5Ac form of sialic acid <i>versus</i> those that contain Neu5Gc. We propose using this approach to evaluate sialidase preferences for diverse potential substrates

Enhanced Cross-Linking of Diazirine-Modified Sialylated Glycoproteins Enabled through Profiling of Sialidase Specificities

Sialic-acid-mediated interactions play critical roles on the cell surface, providing an impetus for the development of methods to study this important monosaccharide. In particular, photo-cross-linking sialic acids incorporated onto cell surfaces have allowed covalent capture of transient interactions between sialic acids and sialic-acid-recognizing proteins via cross-linking. However, natural sialic acids also present on the cell surface compete with photo-cross-linking sialic acids in binding events, limiting cross-linking yields. In order to improve the utility of one such photo-cross-linking sialic acid, SiaDAz, we examined a number of sialidases, enzymes that remove sialic acids from glycoconjugates, to find one that would cleave natural sialic acids but remain inactive toward SiaDAz. Using this sialidase, we improved SiaDAz-mediated cross-linking of an antisialyl Lewis X antibody and of endoglin. This protocol can be applied generally to sialic-acid-mediated interactions and will facilitate identification of sialic acid binding partners

Conditional Glycosylation in Eukaryotic Cells Using a Biocompatible Chemical Inducer of Dimerization

Conditional Glycosylation in Eukaryotic Cells Using a Biocompatible Chemical Inducer of Dimerizatio

<i>T</i>. <i>brucei</i> pyrimidine pathway.

<p>Green lines salvage routes, blue lines <i>de novo</i> pathway, black lines interconversion routes, and the red dotted line indicates a reaction that is not present in trypanosomatids. The numbers above each arrow represent the enzyme catalyzing the reaction (EC number): <b>1–6</b>: carbamoyl phosphate synthase (6.3.5.5), aspartate carbamoyl transferase (2.1.3.2), dihydroorotase (3.5.2.3), dihydroorotate dehydrogenase (1.3.98.1), orotate phosphoribosyltransferase (2.4.2.10), orotidine 5-phosphate decarboxylase (4.1.1.23); <b>7</b> UMP-CMP kinase (2.7.4.14); <b>8</b>: nucleoside diphosphatase (3.6.1.6); <b>9</b>: nucleoside diphosphate kinase (2.7.4.6); <b>10</b>: cytidine triphosphate synthase (6.3.4.2); <b>11</b>: ribonucleoside diphosphate reductase (1.17.4.1); <b>12</b>: thymidylate kinase (2.7.4.9); <b>13</b>: deoxyuridine triphosphatase (dUTPase) (3.6.1.23); <b>14</b>: dihydrofolate reductase-thymidylate synthase (2.1.1.45); <b>15</b>:cytidine deaminase (CDA) (3.5.4.5); <b>16</b>: thymidine kinase (TK)(2.7.1.21); <b>17</b>: uridine phosphorylase (2.4.2.3); <b>18</b>:uracil phosphoribosyltransferase (2.4.2.9); 19: HD-domain 5’-nucleotidase (3.1.3.89); <b>20</b>: UDP-glucose pyrophosphorylase (2.7.7.9); <b>21</b>: UTP N-acetyl-α-D-glucosamine-1-phosphate uridylyltransferase (2.7.7.23); <b>22</b>: UDP-glucose 4-epimerase (5.1.3.2). The pathway was constructed based on the annotation described in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006010#ppat.1006010.ref010" target="_blank">10</a>] and modified to incorporate results from our studies. Additionally enzyme <b>7</b> was added based on the published report that one of seven encoded adenylate kinases (ADKG) was biochemically characterized and shown to be a UMP-CMP kinase [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006010#ppat.1006010.ref016" target="_blank">16</a>].</p

<i>Hs</i>DCTD rescues the growth defect in <i>Tb</i>TK RNAi and <i>Tb</i>TK null cell lines.

<p>A. Growth curves for <i>TK</i> RNAi cells or <i>TK</i> RNAi cells containing a Tet-regulated expression plasmid for <i>Hs</i>DCTD. Cell growth was monitored ±Tet for the indicated days. Error bars represent SD for triplicate biological replicates. Inset shows a Western blot of <i>Hs</i>DCTD expression ± Tet at 48 h. B. qPCR analysis of <i>Tb</i>TK mRNA expression in both the <i>TK</i> RNAi cell line and the <i>TK</i> RNAi <i>Hs</i>DCTD rescue line (±Tet 48 h). Error bars represent SEM for triplicate data. Data were normalized to the -Tet control, which is in the background of the single allele TK knockout. C. Growth analysis of <i>TK</i> null cells expressing either FLAG-tagged <i>Tb</i>TK (c-null) or FLAG-tagged <i>Hs</i>DCTD under the control of the Tet promoter. Error bars represent SD for triplicate biological replicates. Inset shows western blot analysis of the <i>Hs</i>DCTD <i>TK</i> null cells 2 days and 5 days after Tet withdraw.</p

Catalytically active TK is required to rescue the <i>TK</i> RNAi growth phenotype.

<p>A-B. Growth analysis of TK RNAi cells (±Tet) expressing <i>Tb</i>TK or <i>Hs</i>TK under Tet control. Cell growth was monitored for the indicated days. Error bars represent SD for triplicate biological replicates. C. qPCR analysis of <i>Tb</i>TK mRNA levels in <i>TK</i> RNAi knockdown cells in the absence and presence of the <i>Hs</i>TK rescue plasmid 48 h after Tet addition. Error bars represent SEM for triplicate data. Data were normalized to TK levels in wild-type SM cells. D-E. Growth analysis of <i>TK</i> RNAi cells (±Tet) expressing active-site mutant TK enzymes, <i>Tb</i>TK E286A or <i>Hs</i>TK K32I under Tet control. Error bars represent SD for triplicate biological replicates. Insets show western blots of the AU1-<i>Tb</i>TK (A), FLAG-<i>Tb</i>TK (D) or FLAG-<i>Hs</i>TK (B,E) rescued RNAi lines comparing ±Tet for 48 h, though in panel E, <i>Hs</i>TK K32I was detected with a <i>Hs</i>TK antibody. <i>Tb</i>BiP was detected as a loading control.</p

Venn diagram showing the distribution of TK, DCTD and dCTP deaminase in representative protists and higher eukaryotes.

<p>The overlapping regions represent organisms that possess both of the indicated genes.</p

TK is essential for <i>in vitro</i> growth and infectivity in mice.

<p>A. Growth analysis of <i>TK</i> c-null cells and wild-type SM cells ±Tet. Expression of ectopic FLAG-tagged <i>Tb</i>TK is under Tet control, thus removal of Tet leads to loss of <i>Tb</i>TK expression. Cells were grown in HMI-19 medium supplemented with either normal serum (NS) or dialyzed serum (DS). Cell growth was monitored for the indicated days. Error bars represent standard deviation (SD) for triplicate biological replicates. Inset shows western blot analysis of FLAG-tagged <i>Tb</i>TK expression ±Tet for 24h. <i>Tb</i>BiP was detected as a loading control. B. qPCR analysis comparing mRNA expression levels of <i>Tb</i>TK to the TERT control ±Tet for 24 h. Error bars represent standard error of the mean (SEM) for triplicate data. C. Survival analysis of wild-type SM and TK c-null infected mice (±Dox) 1–30 days post infection for three mice per group.</p

Steady-state kinetic analysis of <i>T</i>. <i>brucei</i> HD domain 5’-nucleotidase.

<p>A. Metal ion dependence. dCMP (1 mM) was used as the substrate and metal concentrations are noted on the figure. B. Substrate preference. Substrate concentrations were 1 mM and these assays were run in the presence of 0.5 mM Co⁺². The < symbol on the graph indicates that the activity was below the level of detection. Data were collected in triplicate and error bars represent the SD of the mean.</p