Active-site peptide "fingerprinting" of glycosidases in complex mixtures by mass spectrometry. Discovery of a novel retaining beta-1,4-glycanase in Cellulomonas fimi

New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining beta-endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining beta-1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi beta-1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares approximately 70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases.     

The expression of Cellulomonas fimi cellulase genes in Brevibacterium lactofermentum

The exoglucanase gene (cex) and the endoglucanase A gene (cenA) from Cellulomonas fimi were subcloned into the Escherichia coli/Brevibacterium lactofermentum shuttle vector pBK10. Both genes were expressed to five to ten times higher levels in B. lactofermentum than in E. coli, probably because these genes were expressed from C. fimi promoters. In B. lactofermentum virtually all of the enzyme activities were in the culture supernatant. This system will facilitate analysis of the expression of the C. fimi genes in and secretion of their products from a Gram-positive bacterium.     

Production of bioactive cellulose films reconstituted from ionic liquids

A new method for introducing enzymes into cellulosic matrixes which can be formed into membranes, films, or beads has been developed using a cellulose-in-ionic-liquid dissolution and regeneration process. Initial results on the formation of thin cellulose films incorporating dispersed laccase indicate that active enzyme-encapsulated films can be prepared using this methodology and that precoating the enzyme with a second, hydrophobic ionic liquid prior to dispersion in the cellulose/ionic liquid solution can provide an increase in enzyme activity relative to that of untreated films, presumably by providing a stabilizing microenvironment for the enzyme.     

Expression and Secretion of a Cellulomonas fimi Exoglucanase in Saccharomyces cerevisiae

We used the yeast MEL1 gene for secreted alpha-galactosidase to construct cartridges for the regulated expression of foreign proteins from Saccharomyces cerevisiae. The gene for a Cellulomonas fimi beta-1,4-exoglucanase was inserted into one cartridge to create a fusion of the alpha-galactosidase signal peptide to the exoglucanase. Yeast transformed with plasmids containing this construction produced active extracellular exoglucanase when grown under conditions appropriate to MEL1 promoter function. The cells also produced active intracellular enzyme. The secreted exoglucanase was N-glycosylated and was produced continuously during culture growth. It hydrolyzed xylan, carboxymethyl cellulose, 4-methylumbelliferyl-beta-d-cellobiose, and p-nitrophenyl-beta-d-cellobiose. A comparison of the recombinant S. cerevisiae enzyme with the native C. fimi enzyme showed the yeast version to have an identical K(m) and pH optimum but to be more thermostable.     

Mannan-degrading enzymes from Cellulomonas fimi.

The genes man26a and man2A from Cellulomonas fimi encode mannanase 26A (Man26A) and beta-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced by Escherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian beta-mannosidases.     

Nucleotide sequence of the endoglucanase C gene (cenC) of Cellulomonas fimi, its high-level expression in Escherichia coli, and characterization of its products

The cenC gene of Cellulomonas fimi, encoding endoglucanase CenC, has an open reading frame of 1101 codons closely followed by a 9 bp inverted repeat. The predicted amino acid sequence of mature CenC, which is 1069 amino acids long, is very unusual in that it has a 150-amino-acid tandem repeat at the N-terminus and an unrelated 100-amino-acid tandem repeat at the C-terminus. CenC belongs to subfamily E1 of the beta-1,4-glycanases. High-level expression in Escherichia coli of cenC from a 3.6 kbp fragment of C. fimi DNA leads to levels of CenC which exceed 10% of total cell protein. Most of the CenC is in the cytoplasm in an inactive form. About 60% of the active fraction of CenC is in the periplasm. The catalytic properties of the active CenC are indistinguishable from those of native CenC from C. fimi. The Mr of CenC from E. coli and C. fimi is approximately 130 kDa. E. coli and C. fimi also produce an endoglucanase, CenC', of approximate Mr 120kDa and with the same N-terminal amino acid sequence and catalytic properties as CenC. CenC' appears to be a proteolytic product of CenC. CenC and CenC' can bind to cellulose and to Sephadex. CenC is the most active component of the C. fimi cellulase system isolated to date.     

Immunofluorometric activity-based probe analysis of active KLK6 in biological fluids

Immunoassay measurements of human kallikrein-related peptidases (KLKs) such as prostate-specific antigen (KLK3) are of great value as diagnostic indices of cancer. Despite extensive knowledge of the abundance of immunoreactive KLKs in normal and cancer-related settings, there is little information available about the proportion of immunoreactive KLK that represents active enzyme in such samples. Using KLK6 as a prototype enzyme, we have developed an assay using a serine proteinase-targeted activity-based probe coupled to antibody capture. By employing activity-based labeling, we were able to quantify the proportion of enzymatically active relative to total immunoreactive KLK6 in crude cerebrospinal fluid from routine analyses and ascites fluid from ovarian cancer patients, as well as in supernatants from cancer cell lines. Our approach allowed monitoring of pro-KLK6 conversion to its active enzyme species and demonstrated that up to 5% of immunoreactive KLK6 detected in clinical samples represents active enzyme. We suggest that this new activity-based probe assay will prove of value as a complement to routine KLK immunoassay measurements for validating KLKs as cancer biomarkers.     

Silanediol peptidomimetics. Evaluation of four diastereomeric ACE inhibitors

Four diastereomers of a Phe-Ala peptide mimic incorporating a central silanediol group have been individually prepared and tested as inhibitors of angiotensin-converting enzyme (ACE). Three of the silanediols exhibit levels of inhibition that are similar to those of corresponding ketones reported by Almquist. For the fourth diastereomer, with both stereogenic carbons inverted relative to the most active isomer, the ketone gives the least enzyme inhibition whereas the silanediol shows a surprisingly low IC50 value.     

Trifunctional chemical probes for the consolidated detection and identification of enzyme activities from complex proteomes

Chemical probes that covalently modify the active sites of enzymes in complex proteomes are useful tools for identifying enzyme activities associated with discrete (patho) physiological states. Researchers in proteomics typically use two types of activity-based probes to fulfill complementary objectives: fluorescent probes for rapid and sensitive target detection and biotinylated probes for target purification and identification. Accordingly we hypothesized that a strategy in which the target detection and target isolation steps of activity-based proteomic experiments were merged might accelerate the characterization of differentially expressed protein activities. Here we report the synthesis and application of trifunctional chemical proteomic probes in which elements for both target detection (e.g. rhodamine) and isolation (e.g. biotin) are appended to a sulfonate ester reactive group, permitting the consolidated visualization and affinity purification of labeled proteins by a combination of in-gel fluorescence and avidin chromatography procedures. A trifunctional phenyl sulfonate probe was used to identify several technically challenging protein targets, including the integral membrane enzyme 3beta-hydroxysteroid dehydrogenase/Delta5-isomerase and the cofactor-dependent enzymes platelet-type phosphofructokinase and type II tissue transglutaminase. The latter two enzyme activities were significantly up-regulated in the invasive estrogen receptor-negative (ER(-)) human breast cancer cell line MDA-MB-231 relative to the non-invasive ER(+) breast cancer lines MCF7 and T-47D. Collectively these studies demonstrate that chemical proteomic probes incorporating elements for both target detection and target isolation fortify the important link between the visualization of differentially expressed enzyme activities and their subsequent molecular identification, thereby augmenting the information content achieved in activity-based profiling experiments.     

The structure and characterization of a modular endo-beta-1,4-mannanase from Cellulomonas fimi

The endo-beta-1,4-mannanase from the soil bacterium Cellulomonas fimi is a modular plant cell wall degrading enzyme involved in the hydrolysis of the backbone of mannan, one of the most abundant polysaccharides of the hemicellulosic network in the plant cell wall. The crystal structure of a recombinant truncated endo-beta-1,4-mannanase from C. fimi (CfMan26A-50K) was determined by X-ray crystallography to 2.25 A resolution using the molecular replacement technique. The overall structure of the enzyme consists of a core (beta/alpha)8-barrel catalytic module characteristic of clan GH-A, connected via a linker to an immunoglobulin-like module of unknown function. A complex with the oligosaccharide mannotriose to 2.9 A resolution has also been obtained. Both the native structure and the complex show a cacodylate ion bound at the -1 subsite, while subsites -2, -3, and -4 are occupied by mannotriose in the complex. Enzyme kinetic analysis and the analysis of hydrolysis products from manno-oligosaccharides and mannopentitol suggest five important active-site cleft subsites. CfMan26A-50K has a high affinity -3 subsite with Phe325 as an aromatic platform, which explains the mannose releasing property of the enzyme. Structural differences with the homologous Cellvibrio japonicus beta-1,4-mannanase (CjMan26A) at the -2 and -3 subsites may explain the poor performance of CfMan26A mutants as "glycosynthases".     

Activity-based probes as a tool for functional proteomic analysis of proteases

Traditional proteomics methodology allows global analysis of protein abundance but does not provide information on the regulation of protein activity. Proteases, in particular, are known for their multilayered post-translational activity regulation that can lead to a significant difference between protease abundance levels and their enzyme activity. To address these issues, the field of activity-based proteomics has been established in order to characterize protein activity and monitor the functional regulation of enzymes in complex proteomes. In this review, we present structural features of activity-based probes for proteases and discuss their applications in proteomic profiling of various catalytic classes of proteases.     

Activity-based probes as a tool for functional proteomic analysis of proteases

Traditional proteomics methodology allows global analysis of protein abundance but does not provide information on the regulation of protein activity. Proteases, in particular, are known for their multilayered post-translational activity regulation that can lead to a significant difference between protease abundance levels and their enzyme activity. To address these issues, the field of activity-based proteomics has been established in order to characterize protein activity and monitor the functional regulation of enzymes in complex proteomes. In this review, we present structural features of activity-based probes for proteases and discuss their applications in proteomic profiling of various catalytic classes of proteases.     

Glutamic acid 274 is the nucleophile in the active site of a "retaining" exoglucanase from Cellulomonas fimi.

In addition to its known substrate activity with p-nitrophenyl beta-cellobioside, the exoglucanase from Cellulomonas fimi has been shown to utilize substituted phenyl beta-glucosides as substrates, of which the best is 2',4'-dinitrophenyl beta-D-glucopyranoside. The enzyme can be inactivated by treatment with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside, by trapping of the covalent intermediate in catalysis, as has been shown for a beta-glucosidase (Withers, S.G., and Street, I.P. (1988) J. Am. Chem. Soc. 110, 8551-8553). The intermediate formed is stable but can undergo turnover in the presence of cellobiose, reactivating the enzyme by transglycosylation. Using a tritium-labeled inactivator it has been possible to isolate and sequence a radiolabeled peptide from this enzyme, and the active site nucleophile has been identified as glutamic acid residue 274. This glutamic acid residue and its sequentially proximal amino acids are absolutely conserved in the homologous family F of cellulases.     

Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities

We established a novel technique for differential activity-based gel electrophoresis (DABGE) of lipolytic enzymes from two different biological samples. For this purpose, a set of three fluorescent suicide inhibitors was developed. These probes possess the same substrate analogous structures but carry different cyanine dyes (Cy2b, Cy3, and Cy5) as reporter fluorophores. For comparison of enzyme profiles, two samples are individually labeled with a different probe followed by mixing, gel electrophoresis, fluorescence imaging, and identification of the tagged proteins by MS/MS. Protocols for quantitative determination of active enzymes were developed on the basis of lipolytic proteomes that had been admixed with defined amounts of known lipases and esterases. A detailed analysis of the fluorescence intensities showed that the found enzyme ratios very closely reflected the relative amounts of the labeled enzymes that were used for spiking. The DABGE method was used to compare the lipolytic proteomes of brown and white adipose tissue showing specific enzyme patterns of both samples. This study represents the first application of this technology for comparative analysis of lipases and esterases. Further applications of this technique can be expected to provide entirely new information on lipid enzymology in health and disease with high precision.     

Activity-based proteomics: enzymatic activity profiling in complex proteomes

Summary. In the postgenomic era new technologies are emerging for global analysis of protein function. The introduction of active site-directed chemical probes for enzymatic activity profiling in complex mixtures, known as activity-based proteomics has greatly accelerated functional annotation of proteins. Here we review probe design for different enzyme classes including serine hydrolases, cysteine proteases, tyrosine phosphatases, glycosidases, and others. These probes are usually detected by their fluorescent, radioactive or affinity tags and their protein targets are analyzed using established proteomics techniques. Recent developments, such as the design of probes for in vivo analysis of proteomes, as well as microarray technologies for higher throughput screenings of protein specificity and the application of activity-based probes for drug screening are highlighted. We focus on biological applications of activity-based probes for target and inhibitor discovery and discuss challenges for future development of this field.     

The binding of Cellulomonas fimi endoglucanase C (CenC) to cellulose and Sephadex is mediated by the N-terminal repeats

Endoglucanase C (CenC) from Cellulomonas fimi binds to cellulose and to Sephadex. The enzyme has two contiguous 150-amino-acid repeats (N1 and N2) at its N-terminus and two unrelated contiguous 100-amino-acid repeats (C1 and C2) at its C-terminus. Polypeptides corresponding to N1, N1N2, C1, and C1C2 were produced by expression of appropriate cenC gene fragments in Escherichia coli. N1N2, but not N1 alone, binds to Sephadex; both polypeptides bind to Avicel, (a heterogeneous cellulose preparation containing both crystalline and non-crystalline components). Neither C1 nor C1C2 binds to Avicel or Sephadex. N1N2 and N1 bind to regenerated ('amorphous') cellulose but not to bacterial crystalline cellulose; the cellulose-binding domain of C. fimi exoglucanase Cex binds to both of these forms of cellulose. Amino acid sequence comparison reveals that N1 and N2 are distantly related to the cellulose-binding domains of Cex and C. fimi endoglucanases A and B.     

Rapid divergence in expression between duplicate genes inferred from microarray data

For more than 30 years, expression divergence has been considered as a major reason for retaining duplicated genes in a genome, but how often and how fast duplicate genes diverge in expression has not been studied at the genomic level. Using yeast microarray data, we show that expression divergence between duplicate genes is significantly correlated with their synonymous divergence (K_S) and also with their nonsynonymous divergence (K_A) if K_A ≤ 0.3. Thus, expression divergence increases with evolutionary time, and K_A is initially coupled with expression divergence. More interestingly, a large proportion of duplicate genes have diverged quickly in expression and the vast majority of gene pairs eventually become divergent in expression. Indeed, more than 40% of gene pairs show expression divergence even when K_S is ≤ 0.10, and this proportion becomes >80% for K_S > 1.5. Only a small fraction of ancient gene pairs do not show expression divergence.     

Examination of the carboxylesterase phenotype in human liver

Carboxylesterases (CES) metabolize esters. Two CES isoforms are expressed in human liver (CES1 and CES2) and liver extracts are used in reaction phenotyping studies to discern interindividual metabolic variation. We tested the hypothesis that an individual's CES phenotype can be characterized by reporter substrates/probes that interrogate native CES1 and CES2 activities in liver and immunoblotting methods. We obtained 25 livers and found that CES1 is the main hydrolytic enzyme. Moreover, although CES1 protein levels were similar, we observed large interindividual variation in bioresmethrin hydrolysis rates (17-fold), a pyrethroid metabolized by CES1 but not CES2. Bioresmethrin hydrolysis rates did not correlate with CES1 protein levels. In contrast, procaine hydrolysis rates, a drug metabolized by CES2 but not CES1, were much less variant (3-fold). Using activity-based fluorophosphonate probes (FP-biotin), which covalently reacts with active serine hydrolases, CES1 protein was the most active enzyme in the livers. Finally, using bioorthogonal probes and click chemistry methodology, the half-life of CES 1 and 2 in cultured HepG2 cells was estimated at 96 h. The cause of the differential CES1 activities is unknown, but the underlying factors will be important to understand because several carboxylic acid ester drugs and environmental toxicants are metabolized by this enzyme.     

Evaluation of clinical colon carcinoma using activity-based proteomic profiling

The evaluation of clinical tumor tissues is a valuable approach to discovering novel drug targets because of the direct relevance of human samples. We used activity-based proteomic profiling (ABPP) to study the differences in serine hydrolase activities from 12 matched pairs of clinical normal and tumor colon tissues. Unlike traditional proteomics or measures of mRNA abundance, ABPP actually quantifies enzymatic activities, a characteristic crucial for drug targeting. Several serine hydrolases were differentially active in tumor vs normal tissues, despite a lack of obvious corresponding alterations in protein expression. We identified one tumor-specific activity by mass spectrometry to be fibroblast activation protein (FAP), an integral membrane serine protease that has been reported to be present only in tumor stroma or during wound healing and absent in normal tissues. FAP activity was further found to be approximately twofold higher in stage III relative to stage II colon cancer, suggestive of a role in tumor progression. We were also able to identify other proteins, some of which had not been previously linked to cancer, which had higher activity in tumors. Our results demonstrate the applicability of ABPP for the efficient identification of multiple clinical disease targets.     

Small-molecule probes elucidate global enzyme activity in a proteomic context

The recent dramatic improvements in high-resolution mass spectrometry (MS) have revolutionized the speed and scope of proteomic studies. Conventional MS-based proteomics methodologies allow global protein profiling based on expression levels. Although these techniques are promising, there are numerous biological activities yet to be unveiled, such as the dynamic regulation of enzyme activity. Chemical proteomics is an emerging field that extends these types proteomic profiling. In particular, activity-based protein profiling (ABPP) utilizes small-molecule probes to monitor enzyme activity directly in living intact subjects. In this mini-review, we summarize the unique roles of smallmolecule probes in proteomics studies and highlight some recent examples in which this principle has been applied. [BMB Reports 2014; 47(3): 149-157]