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1.
J Nucl Med ; 65(3): 386-393, 2024 Mar 01.
Article in English | MEDLINE | ID: mdl-38272704

ABSTRACT

Radioimmunoconjugates targeting human epidermal growth factor receptor 2 (HER2) have shown potential to noninvasively visualize HER2-positive tumors. However, the stochastic approach that has been traditionally used to radiolabel these antibodies yields poorly defined and heterogeneous products with suboptimal in vivo performance. Here, we describe a first-in-human PET study on patients with HER2-positive breast cancer evaluating the safety, biodistribution, and dosimetry of 89Zr-site-specific (ss)-pertuzumab PET, a site-specifically labeled radioimmunoconjugate designed to circumvent the limitations of random stochastic lysine labeling. Methods: Six patients with HER2-positive metastatic breast cancer were enrolled in a prospective clinical trial. Pertuzumab was site-specifically modified with desferrioxamine (DFO) via a novel chemoenzymatic strategy and subsequently labeled with 89Zr. Patients were administered 74 MBq of 89Zr-ss-pertuzumab in 20 mg of total antibody intravenously and underwent PET/CT at 1 d, 3-4 d, and 5-8 d after injection. PET imaging, whole-body probe counts, and blood draws were performed to assess the pharmacokinetics, biodistribution, and dosimetry. Results: 89Zr-ss-pertuzumab PET/CT was used to assess HER2 status and heterogeneity to guide biopsy and decide the next line of treatment at progression. The radioimmunoconjugate was able to detect known sites of malignancy, suggesting that these tumor lesions were HER2-positive. The optimal imaging time point was 5-8 d after administration, and no toxicities were observed. Dosimetry estimates from OLINDA showed that the organs receiving the highest doses (mean ± SD) were kidney (1.8 ± 0.5 mGy/MBq), liver (1.7 ± 0.3 mGy/MBq), and heart wall (1.2 ± 0.1 mGy/MBq). The average effective dose for 89Zr-ss-pertuzumab was 0.54 ± 0.03 mSv/MBq, which was comparable to both stochastically lysine-labeled 89Zr-DFO-pertuzumab and 89Zr-DFO-trastuzumab. One patient underwent PET/CT with both 89Zr-ss-pertuzumab and 89Zr-DFO-pertuzumab 1 mo apart, with 89Zr-ss-pertuzumab demonstrating improved lesion detection and higher tracer avidity. Conclusion: This study demonstrated the safety, dosimetry, and potential clinical applications of 89Zr-ss-pertuzumab PET/CT. 89Zr-ss-pertuzumab may detect more lesions than 89Zr-DFO-pertuzumab. Potential clinical applications include real-time evaluation of HER2 status to guide biopsy and assist in treatment decisions.


Subject(s)
Breast Neoplasms , Immunoconjugates , Humans , Female , Breast Neoplasms/diagnostic imaging , Breast Neoplasms/drug therapy , Lysine , Positron Emission Tomography Computed Tomography , Prospective Studies , Tissue Distribution , Antibodies, Monoclonal, Humanized/therapeutic use , Immunoconjugates/therapeutic use
2.
Anal Bioanal Chem ; 413(20): 4989-5001, 2021 Aug.
Article in English | MEDLINE | ID: mdl-34231000

ABSTRACT

Antibody drug conjugates (ADCs) represent a rapidly growing modality for the treatment of numerous oncology indications. The complexity of analytical characterization method development is increased due to the potential for synthetic intermediates and process-related impurities. In addition, the cytotoxicity of such materials provides an additional challenge with regard to handling products and/or sharing materials with analytical collaborators and/or vendors for technology development. Herein, we have utilized a site-specific chemoenzymatic glycoconjugation strategy for preparing ADC mimetics composed of the NIST monoclonal antibody (NISTmAb) conjugated to non-cytotoxic payloads representing both small molecules and peptides. The materials were exhaustively characterized with high-resolution mass spectrometry-based approaches to demonstrate the utility of each analytical method for confirming the conjugation fidelity as well as deep characterization of low-abundance synthetic intermediates and impurities arising from payload raw material heterogeneity. These materials therefore represent a widely available test metric to develop novel ADC analytical methods as well as a platform to discuss best practices for extensive characterization.


Subject(s)
Antibodies, Monoclonal/chemistry , Immunoconjugates , Polysaccharides/chemistry , Chromatography, Liquid , Neoplasms/therapy , Peptide Mapping , Tandem Mass Spectrometry
3.
Theranostics ; 9(15): 4409-4420, 2019.
Article in English | MEDLINE | ID: mdl-31285769

ABSTRACT

Antibody-based PET tracers are exceptionally well-suited for determination of the in vivo biodistribution and quantification of therapeutic antibodies. The continued expansion in antibody-based therapeutics has accordingly driven the development towards more robust conjugation strategies in order to reliably predict the performance of such agents. We therefore aimed to evaluate the effect of site-specific labeling by enzymatic remodeling on the stability, immuno-reactivity and tumor-targeting properties of the monoclonal antibody (mAb) trastuzumab and compare it to conventional, random labeling in a HER2-positive xenograft mouse model. Methods: Trastuzumab was conjugated with the p-SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator randomly on lysine residues or site-specifically on enzymatically modified glycans using either ß-galactosidase or endoglycosidase S2 prior to 89Zr radiolabeling. 89Zr-DFO-trastuzumab was injected into SK-OV-3 tumor-bearing NMRI nude mice. The antibody dose was titrated with either 100 µg or 500 µg of unlabeled trastuzumab. Mice underwent small animal PET/CT imaging 24, 70 and 120 hours post-injection for longitudinal assessment. Parallel experiments were conducted with an isotype control matched antibody. In vivo imaging was supported by conventional ex vivo biodistribution and HER2 immuno-histochemistry. Furthermore, site-specifically labeled 89Zr-DFO-trastuzumab was evaluated in a panel of subcutaneous patient-derived xenograft (PDX) models. Additionally, the affinity, in vitro stability and immuno-reactivity were assessed for all tracers. Results: Site-specific labeling significantly increased PET tumor uptake (One-way ANOVA, p<0.0001) at all time-points when compared to random labeling. Mean tumor uptakes were 6.7 ± 1.7, 13.9 ± 3.3 and 15.3 ± 3.8 % injected dose per gram tissue (%ID/g) at 70 hours post-injection, for random, ß-galactosidase or endoglycosidase S2 labeled probes, respectively. Co-injection with unlabeled trastuzumab increased the circulation time of tracers but did not alter tumor uptake notably. Site-specific probes presented with a superior in vitro stability and immuno-reactivity compared to the randomly labeled probe. Ex vivo biodistribution confirmed the data obtained by in vivo PET imaging, and site-specific 89Zr-DFO-trastuzumab successfully detected HER2-positive tumors in PDX mouse models. Conclusion: 89Zr-DFO-trastuzumab is well-matched for specific immuno-PET imaging of HER2-positive tumors and site-specific labeling of trastuzumab by the SiteClickTM technology minimizes the impact of the DFO chelator on immuno-reactivity, stability and biodistribution. These findings support further development of site-specifically radiolabeled mAbs for immuno-PET.


Subject(s)
Deferoxamine/chemistry , Neoplasms/drug therapy , Neoplasms/immunology , Positron-Emission Tomography , Subcutaneous Tissue/metabolism , Trastuzumab/therapeutic use , Xenograft Model Antitumor Assays , Zirconium/chemistry , Animals , Cell Line, Tumor , Humans , Mice , Neoplasms/diagnostic imaging , Receptor, ErbB-2 , Subcutaneous Tissue/pathology , Tissue Distribution
4.
Mol Pharm ; 15(3): 892-898, 2018 03 05.
Article in English | MEDLINE | ID: mdl-29356543

ABSTRACT

The conjugation of antibodies with cytotoxic drugs can alter their in vivo pharmacokinetics. As a result, the careful assessment of the in vivo behavior, and specifically the tumor-targeting properties, of antibody-drug conjugates represents a crucial step in their development. In order to facilitate this process, we have created a methodology that facilitates the dual labeling of an antibody with both a toxin and a radionuclide for positron emission tomography (PET). To minimize the impact of these modifications, this chemoenzymatic approach leverages strain-promoted azide-alkyne click chemistry to graft both cargoes to the heavy chain glycans of the immuoglobulin's Fc domain. As a proof-of-concept, a HER2-targeting trastuzumab immunoconjugate was created bearing both a monomethyl auristatin E (MMAE) toxin as well as the long-lived positron-emitting radiometal 89Zr ( t1/2 ≈ 3.3 days). Both the tumor targeting and therapeutic efficacy of the 89Zr-trastuzumab-MMAE immunoconjugate were validated in vivo using a murine model of HER2-expressing breast cancer. The site-specifically dual-labeled construct enabled the clear visualization of tumor tissue via PET imaging, producing tumoral uptake of ∼70%ID/g. Furthermore, a longitudinal therapy study revealed that the immunoconjugate exerts significant antitumor activity, leading to a >90% reduction in tumor volume over the course of 20 days.


Subject(s)
Antibodies, Monoclonal, Humanized/administration & dosage , Antineoplastic Agents, Immunological/administration & dosage , Breast Neoplasms/drug therapy , Immunoconjugates/administration & dosage , Positron Emission Tomography Computed Tomography/methods , Animals , Antibodies, Monoclonal, Humanized/pharmacokinetics , Antineoplastic Agents, Immunological/pharmacokinetics , Breast Neoplasms/diagnostic imaging , Breast Neoplasms/pathology , Cell Line, Tumor , Click Chemistry , Drug Development , Female , Humans , Immunoconjugates/chemistry , Immunoconjugates/pharmacokinetics , Mice , Mice, Nude , Radiopharmaceuticals/administration & dosage , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/pharmacokinetics , Receptor, ErbB-2/antagonists & inhibitors , Tissue Distribution , Treatment Outcome , Tumor Burden/drug effects , X-Ray Microtomography/methods
5.
Theranostics ; 6(12): 2267-2277, 2016.
Article in English | MEDLINE | ID: mdl-27924162

ABSTRACT

The complementary nature of positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging makes the development of strategies for the multimodal PET/NIRF imaging of cancer a very enticing prospect. Indeed, in the context of colorectal cancer, a single multimodal PET/NIRF imaging agent could be used to stage the disease, identify candidates for surgical intervention, and facilitate the image-guided resection of the disease. While antibodies have proven to be highly effective vectors for the delivery of radioisotopes and fluorophores to malignant tissues, the use of radioimmunoconjugates labeled with long-lived nuclides such as 89Zr poses two important clinical complications: high radiation doses to the patient and the need for significant lag time between imaging and surgery. In vivo pretargeting strategies that decouple the targeting vector from the radioactivity at the time of injection have the potential to circumvent these issues by facilitating the use of positron-emitting radioisotopes with far shorter half-lives. Here, we report the synthesis, characterization, and in vivo validation of a pretargeted strategy for the multimodal PET and NIRF imaging of colorectal carcinoma. This approach is based on the rapid and bioorthogonal ligation between a trans-cyclooctene- and fluorophore-bearing immunoconjugate of the huA33 antibody (huA33-Dye800-TCO) and a 64Cu-labeled tetrazine radioligand (64Cu-Tz-SarAr). In vivo imaging experiments in mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts clearly demonstrate that this approach enables the non-invasive visualization of tumors and the image-guided resection of malignant tissue, all at only a fraction of the radiation dose created by a directly labeled radioimmunoconjugate. Additional in vivo experiments in peritoneal and patient-derived xenograft models of colorectal carcinoma reinforce the efficacy of this methodology and underscore its potential as an innovative and useful clinical tool.


Subject(s)
Antibodies, Neoplasm/administration & dosage , Carcinoma/diagnostic imaging , Colorectal Neoplasms/diagnostic imaging , Copper Radioisotopes/administration & dosage , Fluorescent Dyes/administration & dosage , Optical Imaging/methods , Positron-Emission Tomography/methods , Animals , Antibodies, Neoplasm/chemistry , Copper Radioisotopes/chemistry , Disease Models, Animal , Fluorescent Dyes/chemistry , Heterografts , Mice
6.
Bioconjug Chem ; 27(8): 1789-95, 2016 08 17.
Article in English | MEDLINE | ID: mdl-27356886

ABSTRACT

In recent years, both site-specific bioconjugation techniques and bioorthogonal pretargeting strategies have emerged as exciting technologies with the potential to improve the safety and efficacy of antibody-based nuclear imaging. In the work at hand, we have combined these two approaches to create a pretargeted PET imaging strategy based on the rapid and bioorthogonal inverse electron demand Diels-Alder reaction between a (64)Cu-labeled tetrazine radioligand ((64)Cu-Tz-SarAr) and a site-specifically modified huA33-trans-cyclooctene immunoconjugate ((ss)huA33-PEG12-TCO). A bioconjugation strategy that harnesses enzymatic transformations and strain-promoted azide-alkyne click chemistry was used to site-specifically append PEGylated TCO moieties to the heavy chain glycans of the colorectal cancer-targeting huA33 antibody. Preclinical in vivo validation studies were performed in athymic nude mice bearing A33 antigen-expressing SW1222 human colorectal carcinoma xenografts. To this end, mice were administered (ss)huA33-PEG12-TCO via tail vein injection and-following accumulation intervals of 24 or 48 h-(64)Cu-Tz-SarAr. PET imaging and biodistribution studies reveal that this strategy clearly delineates tumor tissue as early as 1 h post-injection (6.7 ± 1.7%ID/g at 1 h p.i.), producing images with excellent contrast and high tumor-to-background activity concentration ratios (tumor:muscle = 21.5 ± 5.6 at 24 h p.i.). Furthermore, dosimetric calculations illustrate that this pretargeting approach produces only a fraction of the overall effective dose (0.0214 mSv/MBq; 0.079 rem/mCi) of directly labeled radioimmunoconjugates. Ultimately, this method effectively facilitates the high contrast pretargeted PET imaging of colorectal carcinoma using a site-specifically modified immunoconjugate.


Subject(s)
Immunoconjugates/chemistry , Positron-Emission Tomography/methods , Alkynes/chemistry , Animals , Azides/chemistry , Binding Sites , Cell Line, Tumor , Cell Transformation, Neoplastic , Colorectal Neoplasms/diagnostic imaging , Colorectal Neoplasms/pathology , Copper Radioisotopes , Humans , Immunoconjugates/metabolism , Immunoconjugates/pharmacokinetics , Isotope Labeling , Mice , Tissue Distribution
7.
Proc Natl Acad Sci U S A ; 112(52): 15850-5, 2015 Dec 29.
Article in English | MEDLINE | ID: mdl-26668398

ABSTRACT

Molecular imaging agents for preoperative positron emission tomography (PET) and near-infrared fluorescent (NIRF)-guided delineation of surgical margins could greatly enhance the diagnosis, staging, and resection of pancreatic cancer. PET and NIRF optical imaging offer complementary clinical applications, enabling the noninvasive whole-body imaging to localize disease and identification of tumor margins during surgery, respectively. We report the development of PET, NIRF, and dual-modal (PET/NIRF) imaging agents, using 5B1, a fully human monoclonal antibody that targets CA19.9, a well-established pancreatic cancer biomarker. Desferrioxamine (DFO) and/or a NIRF dye (FL) were conjugated to the heavy-chain glycans of 5B1, using a robust and reproducible site-specific (ss) labeling methodology to generate three constructs ((ss)DFO-5B1, (ss)FL-5B1, and (ss)dual-5B1) in which the immunoreactivity was not affected by the conjugation of either label. Each construct was evaluated in a s.c. xenograft model, using CA19.9-positive (BxPC3) and -negative (MIAPaCa-2) human pancreatic cancer cell lines. Each construct showed exceptional uptake and contrast in antigen-positive tumors with negligible nonspecific uptake in antigen-negative tumors. Additionally, the dual-modal construct was evaluated in an orthotopic murine pancreatic cancer model, using the human pancreatic cancer cell line, Suit-2. The (ss)dual-5B1 demonstrated a remarkable capacity to delineate metastases and to map the sentinel lymph nodes via tandem PET-computed tomography (PET/CT) and NIRF imaging. Fluorescence microscopy, histopathology, and autoradiography were performed on representative sections of excised tumors to visualize the distribution of the constructs within the tumors. These imaging tools have tremendous potential for further preclinical research and for clinical translation.


Subject(s)
CA-19-9 Antigen/immunology , Immunoconjugates/immunology , Multimodal Imaging/methods , Pancreatic Neoplasms/metabolism , Positron-Emission Tomography/methods , Spectroscopy, Near-Infrared/methods , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacokinetics , Cell Line, Tumor , Deferoxamine/chemistry , Disease Models, Animal , Female , Fluorescent Dyes/chemistry , Humans , Immunoconjugates/chemistry , Immunoconjugates/pharmacokinetics , Mice, Knockout , Mice, Nude , Microscopy, Fluorescence , Molecular Structure , Pancreatic Neoplasms/diagnosis , Radioisotopes/pharmacokinetics , Reproducibility of Results , Sensitivity and Specificity , Tissue Distribution , Transplantation, Heterologous , Zirconium/chemistry
8.
Proteomics ; 15(12): 2066-77, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25914232

ABSTRACT

Protein acylation plays a critical role in protein localization and function. Acylation is essential for human immunodeficiency virus 1 (HIV-1) assembly and budding of HIV-1 from the plasma membrane in lipid raft microdomains and is mediated by myristoylation of the Gag polyprotein and the copackaging of the envelope protein is facilitated by colocalization mediated by palmitoylation. Since the viral accessory protein NEF has been shown to alter the substrate specificity of myristoyl transferases, and alter cargo trafficking lipid rafts, we hypothesized that HIV-1 infection may alter protein acylation globally. To test this hypothesis, we labeled HIV-1 infected cells with biomimetics of acyl azides, which are incorporated in a manner analogous to natural acyl-Co-A. A terminal azide group allowed us to use a copper catalyzed click chemistry to conjugate the incorporated modifications to a number of substrates to carry out SDS-PAGE, fluorescence microscopy, and enrichment for LC-MS/MS. Using LC-MS/MS, we identified 103 and 174 proteins from the myristic and palmitic azide enrichments, with 27 and 45 proteins respectively that differentiated HIV-1 infected from uninfected cells. This approach has provided us with important insights into HIV-1 biology and is widely applicable to many virological systems.


Subject(s)
Acyl Coenzyme A/metabolism , Biomimetics , HIV Infections/metabolism , HIV-1/physiology , Palmitoyl Coenzyme A/metabolism , Proteome/analysis , Proteomics/methods , Acylation , Acyltransferases/metabolism , Cells, Cultured , Chromatography, Liquid , Click Chemistry , Electrophoresis, Gel, Two-Dimensional , HIV Infections/virology , Humans , Protein Interaction Maps , Proteome/metabolism , Tandem Mass Spectrometry , Viral Proteins/metabolism
9.
Bioconjug Chem ; 25(12): 2123-8, 2014 Dec 17.
Article in English | MEDLINE | ID: mdl-25418333

ABSTRACT

The complementary nature of positron emission tomography (PET) and optical imaging (OI) has fueled increasing interest in the development of multimodal PET/OI probes that can be employed during the diagnosis, staging, and surgical treatment of cancer. Due to their high selectivity and affinity, antibodies have emerged as promising platforms for the development of hybrid PET/OI agents. However, the lack of specificity of many bioconjugation reactions can threaten immunoreactivity and lead to poorly defined constructs. To circumvent this issue, we have developed a chemoenzymatic strategy for the construction of multimodal PET/OI immunoconjugates that have been site-specifically labeled on the heavy chain glycans. The methodology consists of four steps: (1) the enzymatic removal of the terminal galactose residues on the heavy chain glycans; (2) the enzymatic incorporation of azide-bearing galactose (GalNAz) residues into the heavy chain glycans; (3) the strain-promoted click conjugation of chelator- and fluorophore-modified dibenzocyclooctynes to the azide-modified sugars; and (4) the radiolabeling of the immunoconjugate. For proof-of-concept, a model system was created using the colorectal cancer-targeting antibody huA33, the chelator desferrioxamine (DFO), the positron-emitting radiometal (89)Zr, and the near-infrared fluorescent dye Alexa Fluor 680. The bioconjugation strategy is robust and reproducible, reliably producing well-defined and immunoreactive conjugates labeled with (89)Zr, Alexa Fluor 680, or an easily and precisely tuned mixture of the two reporters. In in vivo PET and fluorescence imaging experiments, a hybrid (89)Zr- and Alexa Fluor 680-labeled huA33 conjugate displayed high levels of specific uptake (>45% ID/g) in athymic nude mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts.


Subject(s)
Immunoconjugates/chemistry , Optical Imaging/methods , Positron-Emission Tomography/methods , Animals , Colorectal Neoplasms/diagnosis , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes/chemistry , Galactose/chemistry , Humans , Immunoconjugates/pharmacokinetics , Mice , Multimodal Imaging , Neoplasms, Experimental/diagnosis , Polysaccharides/chemistry , Polysaccharides/immunology , Radioisotopes , Tissue Distribution , Xenograft Model Antitumor Assays , Zirconium
10.
Bioconjug Chem ; 24(6): 1057-67, 2013 Jun 19.
Article in English | MEDLINE | ID: mdl-23688208

ABSTRACT

An enzyme- and click chemistry-mediated methodology for the site-selective radiolabeling of antibodies on the heavy chain glycans has been developed and validated. To this end, a model system based on the prostate specific membrane antigen-targeting antibody J591, the positron-emitting radiometal (89)Zr, and the chelator desferrioxamine has been employed. The methodology consists of four steps: (1) the removal of sugars on the heavy chain region of the antibody to expose terminal N-acetylglucosamine residues; (2) the incorporation of azide-modified N-acetylgalactosamine monosaccharides into the glycans of the antibody; (3) the catalyst-free click conjugation of desferrioxamine-modified dibenzocyclooctynes to the azide-bearing sugars; and (4) the radiolabeling of the chelator-modified antibody with (89)Zr. The site-selective labeling methodology has proven facile, reproducible, and robust, producing (89)Zr-labeled radioimmunoconjguates that display high stability and immunoreactivity in vitro (>95%) in addition to highly selective tumor uptake (67.5 ± 5.0%ID/g) and tumor-to-background contrast in athymic nude mice bearing PSMA-expressing subcutaneous LNCaP xenografts. Ultimately, this strategy could play a critical role in the development of novel well-defined and highly immunoreactive radioimmunoconjugates for both the laboratory and clinic.


Subject(s)
Antibodies/metabolism , Deferoxamine/metabolism , Isotope Labeling , Organometallic Compounds/metabolism , Zirconium/metabolism , beta-Galactosidase/metabolism , Animals , Antibodies/chemistry , Binding Sites , Click Chemistry , Deferoxamine/chemistry , Humans , Male , Mice , Mice, Nude , Molecular Structure , Organometallic Compounds/chemistry , Organometallic Compounds/pharmacokinetics , Polysaccharides/chemistry , Polysaccharides/metabolism , Tumor Cells, Cultured , Zirconium/chemistry , beta-Galactosidase/chemistry
11.
J Proteome Res ; 12(2): 927-36, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23301498

ABSTRACT

The post-translational modification of proteins with N-acetylglucosamine (O-GlcNAc) is involved in the regulation of a wide variety of cellular processes and associated with a number of chronic diseases. Despite its emerging biological significance, the systematic identification of O-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved O-GlcNAc protein enrichment procedure, which exploits metabolic labeling of cells by azide-modified GlcNAc and copper-mediated Click chemistry for purification of modified proteins on an alkyne-resin. On-resin proteolysis using trypsin followed by LC-MS/MS afforded the identification of around 1500 O-GlcNAc proteins from a single cell line. Subsequent elution of covalently resin bound O-GlcNAc peptides using selective ß-elimination enabled the identification of 185 O-GlcNAc modification sites on 80 proteins. To demonstrate the practical utility of the developed approach, we studied the global effects of the O-GlcNAcase inhibitor GlcNAcstatin G on the level of O-GlcNAc modification of cellular proteins. About 200 proteins including several key players involved in the hexosamine signaling pathway showed significantly increased O-GlcNAcylation levels in response to the drug, which further strengthens the link of O-GlcNAc protein modification to cellular nutrient sensing and response.


Subject(s)
Acetylglucosamine/metabolism , Immobilized Proteins/isolation & purification , Immobilized Proteins/metabolism , Peptide Fragments/analysis , Protein Processing, Post-Translational , Proteome/isolation & purification , Proteome/metabolism , Alkynes/chemistry , Azides/chemistry , Chromatography, Liquid , Click Chemistry , Enzyme Inhibitors/pharmacology , HEK293 Cells , Humans , Tandem Mass Spectrometry , Trypsin/chemistry , beta-N-Acetylhexosaminidases/antagonists & inhibitors , beta-N-Acetylhexosaminidases/metabolism
12.
Methods Mol Biol ; 698: 459-84, 2011.
Article in English | MEDLINE | ID: mdl-21431538

ABSTRACT

Human mesenchymal stem cells (hMSCs) are multipotent stem cells that can differentiate into a variety of cell types in vitro including osteoblasts, adipocytes, and chondrocytes. Here we apply a metabolic labeling approach to characterize changes in cellular glycoprotein expression during hMSC differentiation and to identify glycoprotein markers unique to differentiated cell types. The two-step labeling method involves the metabolic incorporation of unnatural azido-modified sugars into protein glycans and subsequent ligation with fluorescent azide-reactive detection probes utilizing the copper (I)-catalyzed cycloaddition reaction between azides and alkynes, or "click" chemistry. Metabolic labeling of cell surface O-linked or sialic acid-containing glycoproteins, or intracellular O-GlcNAc-modified proteins was accomplished by feeding cells the tetraacetylated azide-modified sugar precursors, GalNAz, ManNAz, or GlcNAz, respectively, for 48-72 h prior to harvesting the cells. The cells were then lysed, and protein extracts were reacted with a fluorescent alkyne detection probe. Labeled glycoproteins were analyzed by 1D and 2D gel electrophoresis and detected by fluorescence imaging. Our results demonstrate highly sensitive labeling of O-linked, sialic acid-containing, and O-GlcNAc modified proteins in all cell types without affecting cell growth or morphology. Selective labeling of sialic acid-containing glycoproteins by ManNAz was validated by loss of labeling following digestion with sialidase A. Significant changes in cellular glycoprotein profiles were seen upon differentiation into different cell types, and several putative glycoprotein markers were identified by MALDI peptide fingerprinting. One of these identified proteins, Galectin 1, is validated and shown for the first time to be posttranslationally modified by O-glycosylation, most likely by O-linked N-acetylglucosamine (O-GlcNAc).


Subject(s)
Cell Differentiation , Click Chemistry , Glycoproteins/chemistry , Glycoproteins/metabolism , Mesenchymal Stem Cells/metabolism , Staining and Labeling/methods , Adipocytes/cytology , Adipocytes/metabolism , Alcian Blue/metabolism , Anthraquinones/metabolism , Azo Compounds/metabolism , Biomarkers/chemistry , Biomarkers/metabolism , Blotting, Western , Cell Culture Techniques , Chondrocytes/cytology , Chondrocytes/metabolism , Electrophoresis, Gel, Two-Dimensional , Glycoside Hydrolases/metabolism , Humans , Isoelectric Focusing , Mass Spectrometry , Osteoblasts/cytology , Osteoblasts/metabolism
13.
Electrophoresis ; 30(20): 3598-606, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19784953

ABSTRACT

A recently developed proteomic strategy, the "GG-azide"-labeling approach, is described for the detection and proteomic analysis of geranylgeranylated proteins. This approach involves metabolic incorporation of a synthetic azido-geranylgeranyl analog and chemoselective derivatization of azido-geranylgeranyl-modified proteins by the "click" chemistry, using a tetramethylrhodamine-alkyne. The resulting conjugated proteins can be separated by 1-D or 2-D and pH fractionation, and detected by fluorescence imaging. This method is compatible with downstream LC-MS/MS analysis. Proteomic analysis of conjugated proteins by this approach identified several known geranylgeranylated proteins as well as Rap2c, a novel member of the Ras family. Furthermore, prenylation of progerin in mouse embryonic fibroblast cells was examined using this approach, demonstrating that this strategy can be used to study prenylation of specific proteins. The "GG-azide"-labeling approach provides a new tool for the detection and proteomic analysis of geranylgeranylated proteins, and it can readily be extended to other post-translational modifications.


Subject(s)
Azides/analysis , Protein Prenylation , Proteomics/methods , ras Proteins/analysis , Animals , Azides/metabolism , Cell Line, Tumor , Electrophoresis , Electrophoresis, Gel, Two-Dimensional , Humans , Hydrogen-Ion Concentration , Lamin Type A/metabolism , Mass Spectrometry , Mice , Nuclear Proteins/metabolism , Protein Precursors/metabolism , Rhodamines/chemistry
14.
J Lipid Res ; 50(1): 126-34, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18757838

ABSTRACT

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the synthesis of a truncated prelamin A, commonly called progerin, that contains a carboxyl-terminal farnesyl lipid anchor. The farnesyl lipid anchor helps to target progerin to membrane surfaces at the nuclear rim, where it disrupts the integrity of the nuclear lamina and causes misshapen nuclei. Several lines of evidence have suggested that progerin's farnesyl lipid anchor is crucial for the emergence of disease phenotypes. Because a geranylgeranyl lipid is approximately 45-fold more potent than a farnesyl lipid in anchoring proteins to lipid membranes, we hypothesized that a geranylgeranylated version of progerin might be more potent in eliciting disease phenotypes. To test this hypothesis, we used gene targeting to create mice expressing geranylgeranylated progerin (Lmna(ggHG/+)). We then compared Lmna(ggHG/+) mice, side-by-side, with otherwise identical mice expressing farnesylated progerin (Lmna(HG/+)). Geranylgeranylation of progerin in Lmna(ggHG/+) cells and farnesylation of progerin in Lmna(HG/+) cells was confirmed by metabolic labeling. Contrary to our expectations, Lmna(ggHG/+) mice survived longer than Lmna(HG/+) mice. The Lmna(ggHG/+) mice also exhibited milder bone disease. The steady-state levels of progerin, relative to lamin C, were lower in Lmna(ggHG/+) mice than in Lmna(HG/+) mice, providing a potential explanation for the milder disease in Lmna(ggHG/+) mice.


Subject(s)
Bone Diseases/pathology , Nuclear Proteins/physiology , Progeria/genetics , Protein Precursors/physiology , Animals , Base Sequence , Bone Diseases/metabolism , Genotype , Lamin Type A/metabolism , Lipids/chemistry , Mice , Mice, Transgenic , Models, Biological , Models, Genetic , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Progeria/metabolism , Protein Precursors/genetics , Protein Precursors/metabolism , Syndrome , Tissue Distribution
15.
J Am Chem Soc ; 130(35): 11576-7, 2008 Sep 03.
Article in English | MEDLINE | ID: mdl-18683930

ABSTRACT

We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc.


Subject(s)
Acetylglucosamine/analogs & derivatives , Fluorescent Dyes/chemistry , Proteins/analysis , Acetylglucosamine/analysis , Acetylglucosamine/metabolism , Animals , Biotin/analogs & derivatives , Biotin/chemistry , Cells, Cultured , Fluorescence , Galactosyltransferases/chemistry , Galactosyltransferases/metabolism , Glycosylation , HeLa Cells , Humans , Neurons/chemistry , Neurons/metabolism , Proteins/metabolism , Proteomics/methods , Rats
16.
Biotechniques ; 44(7): 927-9, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18533904

ABSTRACT

The 5-bromo-2'-deoxyuridine (BrdU) labeling of cells followed by antibody staining has been the standard method for direct measurement of cells in the S-phase. Described is an improved method for the detection of S-phase cell cycle progression based upon the application of click chemistry, the copper(I)-catalyzed variant of the Huisgen [3+2] cycloaddition between a terminal alkyne and an azide. 5-ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that is incorporated into DNA during active DNA synthesis, just like BrdU. While the BrdU assay requires harsh chemical or enzymatic disruption of helical DNA structure to allow for direct measurement of cells in the S-phase by the anti-BrdU antibody, the EdU method does not. Elimination of this requirement results in the preservation of helical DNA structure and other cell surface epitopes, decreased assay time, and increased reproducibility.


Subject(s)
Antibodies/immunology , Bromodeoxyuridine/analysis , Deoxyuridine/analogs & derivatives , S Phase , Bromodeoxyuridine/immunology , Bromodeoxyuridine/metabolism , DNA/biosynthesis , Deoxyuridine/metabolism , Flow Cytometry , Humans , Jurkat Cells
17.
Proteomics ; 4(11): 3464-7, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15378738

ABSTRACT

The Multiplexed Proteomics (MP) technology is a new approach that permits quantitative, multicolor fluorescence detection of proteins in one-dimensional or two-dimensional gels. This methodology allows for multiplexed identification and differential analysis of phosphoproteins, glycoproteins, and total proteins within a single gel electrophoresis experiment. Here the MP system was applied to the differential proteomic analysis of pregnancy-induced refractoriness to breast cancer using a rat model system. Differential analyses identified multiple proteins with altered phosphorylation, glycosylation, or protein expression patterns.


Subject(s)
Breast Neoplasms/metabolism , Proteins/metabolism , Proteomics , Animals , Disease Models, Animal , Electrophoresis, Gel, Two-Dimensional , Female , Glycosylation , Phosphoproteins/metabolism , Phosphorylation , Rats
18.
Electrophoresis ; 25(15): 2478-85, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15300765

ABSTRACT

A new solid-phase, fluorescence-based protein assay was developed that quantifies proteins in the presence of detergents, urea and reducing agents (one-dimensional sodium dodecyl sulfate (1-D SDS) lysis buffers and urea isoelectric focusing (IEF) buffers). A specially designed 96-well microplate facilitates application of protein samples to the assay paper and allows easy quantitation of samples using fluorescence microplate readers (top or bottom reading format). Alternatively, stained membranes may be directly scanned using a variety of different laser or charge-coupled device (CCD)-based imaging devices with UV or visible imaging capabilities. Since protein is specifically bound to the membrane, contaminants are readily washed away, avoiding interference with the protein measurement. The protein assay has a dynamic range extending from 10 ng to 5 microg of protein per microliter and requires only 1 microL of sample, which is ideal for samples destined for electrophoresis. The protein-to-protein variability of staining of ten different proteins was determined to be comparable with that of the bicinchoninic acid (BCA) and Lowry assays (16%). Additionally, the quality of the assay according to Z-factor analyses is excellent.


Subject(s)
Detergents/chemistry , Fluorescent Dyes/chemistry , Proteins/chemistry , Quinolines/chemistry , Reducing Agents/chemistry , Animals , Electrophoresis , Humans , Isoelectric Focusing , Sodium Dodecyl Sulfate/chemistry , Urea/chemistry
19.
Proteomics ; 3(7): 1128-44, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12872214

ABSTRACT

Systematic parallel analysis of the phosphorylation status of networks of interacting proteins involved in the regulatory circuitry of cells and tissues is certain to drive research in the post-genomics era for many years to come. Reversible protein phosphorylation plays a critical regulatory role in a multitude of cellular processes, including alterations in signal transduction pathways related to oncogene and tumor suppressor gene products in cancer. While fluorescence detection methods are likely to offer the best solution to global protein quantitation in proteomics, to date, there has been no satisfactory method for the specific and reversible fluorescent detection of gel-separated phosphoproteins from complex samples. The newly developed Pro-Q Diamond phosphoprotein dye technology is suitable for the fluorescent detection of phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins directly in sodium dodecyl sulfate (SDS)-polyacrylamide gels and two-dimensional (2-D) gels. Additionally, the technology is appropriate for the determination of protein kinase and phosphatase substrate preference. Other macromolecules, such as DNA, RNA, and sulfated glycans, fail to be detected with Pro-Q Diamond dye. The staining procedure is rapid, simple to perform, readily reversible and fully compatible with modern microchemical analysis procedures, such as matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Pro-Q Diamond dye technology can detect as little as 1-2 ng of beta-casein, a pentaphosphorylated protein, and 8 ng of pepsin, a monophosphorylated protein. Fluorescence signal intensity correlates with the number of phosphorylated residues on the protein. Through combination of Pro-Q Diamond phosphoprotein stain with SYPRO(R) Ruby protein gel stain, Multiplexed Proteomics technology permits quantitative, dichromatic fluorescence detection of proteins in 2-D gels. This evolving discovery platform allows the parallel determination of protein expression level changes and altered post-translational modification patterns within a single 2-D gel experiment. The linear responses of the fluorescence dyes utilized, allow rigorous quantitation of changes over an unprecedented 500-1000-fold concentration range.


Subject(s)
Phosphoproteins/chemistry , Proteome/chemistry , Animals , Blotting, Western , Catalysis , Cattle , Chickens , DNA/chemistry , Dose-Response Relationship, Drug , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes/pharmacology , HSP90 Heat-Shock Proteins/chemistry , Kinetics , Mass Spectrometry , Oligonucleotide Array Sequence Analysis , Peptides/chemistry , Phosphates/chemistry , Phosphorylation , Protein Kinase C/chemistry , Protein Kinase C-alpha , Proteins/chemistry , RNA/chemistry , Recombinant Proteins/chemistry , Signal Transduction , Spectrometry, Fluorescence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Swine
20.
Int J Toxicol ; 22(1): 9-23, 2003.
Article in English | MEDLINE | ID: mdl-12573945

ABSTRACT

Archaeosomes, liposomes made from polar ether lipids of archaea, show promise for vaccine and drug delivery applications. The potential toxicity of intravenously (14, 70, or 140 mg/kg/day for 5 consecutive days) and orally (gavaged at 55, 275, or 550 mg/kg/day for 10 consecutive days) administered unilamellar archaeosomes, prepared from the total polar lipids (TPLs) extracted from several species of archaea, was assessed in female BALB/c mice. Liposomes prepared from an ester phospholipid composition were included for comparative purposes. Control groups of mice were administered 0.1 ml phosphate-buffered saline (PBS) by either route. Animals were monitored at least once daily for temperature, body weight, and clinical signs of adverse reactions. One day after the last dose, the mice were sacrificed. Blood was collected for selected biochemical/enzyme analyses, and the major organs (heart, lungs, liver, spleen, kidneys) were weighed and examined macroscopically. In addition, the spleens were examined histologically. At the two lower dosages of intravenously administered vesicles, there were no significant indications of toxicity, as compared with the PBS-administered control group. At the highest intravenous dose of 140 mg/kg/day, archaeosomes prepared from the TPL of the extreme halophiles, Halobacterium salinarum and Natronobacterium magadii, indicated potential toxicity, as evidenced by clinical signs (hyperactivity and/or piloerection), drop in body temperature, and loss in body weight. Spleens from mice administered some archaeosomes types, primarily at the highest intravenous dose tested, were enlarged, had increased organ weight, and microscopic examination revealed mild to moderate expansion of the red pulp with increased numbers of hematopoietic cells, but no changes in the white pulp. There were similar clinical signs at one or more of the higher oral doses of the ester liposomes and some of the archaeosome types; however, no other apparent toxicity was observed. Based on this limited mouse study, archaeosomes were generally well tolerated after intravenous or oral delivery at the dosages so indicated in this study.


Subject(s)
Archaea/chemistry , Lipids/toxicity , Liposomes/toxicity , Administration, Oral , Alanine Transaminase/metabolism , Alkaline Phosphatase/metabolism , Animals , Aspartate Aminotransferases/metabolism , Blood Glucose/drug effects , Blood Glucose/metabolism , Blood Urea Nitrogen , Body Weight/drug effects , Dose-Response Relationship, Drug , Female , Heart/drug effects , Injections, Intravenous , Kidney/drug effects , Lipids/administration & dosage , Lipids/blood , Liposomes/administration & dosage , Liposomes/blood , Liver/drug effects , Lung/drug effects , Mice , Mice, Inbred BALB C , Organ Size/drug effects , Temperature , Time Factors
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