Downregulation of the antioxidant protein peroxiredoxin 2 contributes to angiotensin II-mediated podocyte apoptosis.

Podocytes have a significant role in establishing selective permeability of the glomerular filtration barrier. Sustained renin-angiotensin-aldosterone system activation is crucial to the pathogenesis of podocyte injury, but the mechanisms by which angiotensin II modulates podocyte survival due to physiological or injurious stimuli remain unclear. Here, we used proteomic analysis to find new mediators of angiotensin II-induced podocyte injury. Antioxidant protein peroxiredoxin 2 expression was decreased in cultured podocytes stimulated with angiotensin II. Peroxiredoxin 2 was found to be expressed in podocytes in vivo, and its expression was decreased in the glomeruli of rats transgenic for angiotensin II type 1 receptors in a podocyte-specific manner, or in rats infused with angiotensin II. Downregulation of peroxiredoxin 2 in podocytes resulted in increased reactive oxygen species release, protein overoxidation, and inhibition of the Akt pathway. Both treatment with angiotensin II and downregulation of peroxiredoxin 2 expression led to apoptosis of podocytes. Thus, peroxiredoxin 2 is an important modulator of angiotensin II-induced podocyte injury.

Cell type-specific and site directed N-glycosylation pattern of FcγRIIIa.

Human leukocyte receptor IIIa (hFcγRIIIa) plays a prominent role in the elimination of tumor cells by antibody-based cancer therapies. In previous studies, a major impact of the presence of carbohydrates at Asn-162 on the binding between the receptor and the Fc part of wild type fucosylated or glycoengineered nonfucosylated antibodies has been shown. In this study, we performed a site directed carbohydrate analysis at hFcγRIIIa derived from human embryonic kidney (HEK) and Chinese hamster ovary (CHO) cells, respectively. Using mass spectrometry (MS) and a multienzyme protein digest, we analyzed the proteolysis-generated glycopeptides in detail. We could show that hFcγRIIIa expressed by HEK cells was mostly bearing multifucosylated biantennary Asn162-glycans with a major fraction terminating with GalNAc residues replacing the more common Gal. We could demonstrate that the glycan antennae with terminal GalNAc could be sialylated as indicated by a novel reporter ion HexNAcHexNAcNeuAc(+) (m/z 698.28) using a source induced dissociation (SID) scan in the MS cycle. In contrast to the hFcγRIIIa Asn-162 glycosylation pattern from HEK cells, the CHO cells derived receptor contains bi- and triantennary galactosylated and highly sialylated carbohydrates. Our data suggest that the type of expression host system was a dominating factor for formation of distinct glycopatterns of hFcγRIIIa, while the protein sequence and the site of glycosylation remained unchanged for both types of cells. Using surface plasmon resonance (SPR) interaction analysis, we show that the cell type and site specific glycosylation pattern of hFcγRIIIa influences its binding behavior to immunoglobulin molecules.

Differences in CD75s- and iso-CD75s-ganglioside content and altered mRNA expression of sialyltransferases ST6GAL1 and ST3GAL6 in human hepatocellular carcinomas and nontumoral liver tissues.

The sialic acid-specific cytotoxic lectin viscumin and its recombinant equivalent rViscumin specifically bind to CD75s-gangliosides with terminal Neu5Acα6Galß4GlcNAc sequence. We, therefore, comparatively analyzed the content of CD75s-gangliosides and closely related iso-CD75s-gangliosides (terminated by Neu5Acα3Galß4GlcNAc sequence) and the gene expression of associated ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) and ß-galactoside α-2,3-sialyltransferase 6 (ST3GAL6), respectively, in 35 hepatocellular carcinoma (HCC) patients. Ganglioside structures were identified in lipid extracts of matched pairs of malignant and nonmalignant liver tissues by thin-layer chromatography immunodetection coupled with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. CD75s- and iso-CD75s-gangliosides were found to be deregulated in tumor tissues and showed an elevated occurrence in 35 and 41% of HCCs, respectively, compared with nontumoral liver tissues. Statistical analysis revealed a correlation between enhanced iso-CD75s-ganglioside amount and a poor histopathological differentiation (τ = 0.317, P = 0.045) and a significant association of CD75s- and iso-CD75s-ganglioside levels in nontumorous (τ = 0.392, P = 0.003) and in tumorous tissues (τ = 0.650, P < 0.001). Quantitative real-time polymerase chain reaction gene expression analysis of sialyltransferases exhibited no difference in ST6GAL1 expression in cancerous and adjacent noncancerous tissues. Interestingly, the ST3GAL6 expression was significantly diminished in HCCs (P = 0.003). The results indicate that the occurrence of CD75s- and iso-CD75s-gangliosides in tumor tissues is largely independent of the transcriptional expression of ST6GAL1 and ST3GAL6, respectively. Thus, further experiments are required to explore the rationale behind the differential ganglioside level and to validate the applicability of CD75s- and iso-CD75s-gangliosides as targets for individual HCC therapies.

Discovery of a novel unfolded protein response phenotype of cancer stem/progenitor cells from the bone marrow of breast cancer patients.

Metastases arise from disseminated tumor cells (DTC) that colonize secondary organs. However, DTC survival strategies to start metastatic outgrowth are unclear. The hostile (hypoxic, hypoglycemic) microenvironmental conditions of the bone marrow serve as an ideal model environment for investigation of DTC survival strategies under environmental stress. We investigated the breast cancer DTC cell line BC-M1 established from the bone marrow of a cancer patient by 2-D DIGE and MS analysis. We observed specific overexpression of the unfolded protein response (UPR) proteins Grp78, Grp94, and protein disulfide-isomerase in breast, lung, and prostate cancer DTC cell lines from the bone marrow. The UPR contributes to survival under adverse environmental conditions including chemotherapy. We show in cellular models that Grp78 expression of the UPR is regulated by tyrosine 1248 of ErbB-2. The breast cancer DTC cell lines shared stem/progenitor cell cancer phenotypes (CD44(high)/CD24(low)). Immunocytochemical staining of bone marrow samples from breast cancer patients confirmed in situ high expression of Grp78 and Grp94 in DTC of breast cancer patients, indicating the potential of both proteins as novel markers for DTC detection. Our results suggest the presence of a previously not recognized stress resistant DTC population that combines stem/progenitor attributes with an UPR phenotype.

Separation and identification of GM1b pathway Neu5Ac- and Neu5Gc gangliosides by on-line nanoHPLC-QToF MS and tandem MS: toward glycolipidomics screening of animal cell lines.

Monosialoganglioside fraction of YAC-1 lymphoma cells was comprehensively analyzed and structurally defined by nano-high-performance liquid chromatography (nanoHPLC) in on-line conjunction with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF MS). An efficient separation and sensitive detection of Neu5Gc-containing gangliosides from Neu5Ac-containing analogues was for the first time accomplished in a single nanoHPLC/ESI-QTOF MS run, as demonstrated for mouse hybridoma cell GM3 fraction containing GM3(Neu5Ac) and GM3(Neu5Gc) species and further applied for the analysis of YAC-1 lymphoma cell monosialoganglioside fraction. New insights into YAC-1 monosialoganglioside mixture heterogeneity were obtained: 31 distinct species, comprising 18 Neu5Gc-containing gangliosides and 13 Neu5Ac-containing species of GM1b and GalNAc-GM1b type were found to be expressed by YAC-1 cell line. On-line structural elucidation of individually separated Neu5Ac- and Neu5Gc-containing gangliosides provided strong evidence on the "GM1b-pathway" sourcing for monosialoganglioside synthesis. Such an analytical method is documented as superior to the classical approaches by increased speed of analysis, sensitivity and level of information, being thus a viable glycolipidomic tool.

Structural profiling of individual glycosphingolipids in a single thin-layer chromatogram by multiple sequential immunodetection matched with Direct IR-MALDI-o-TOF mass spectrometry.

The thin-layer chromatography (TLC) immunoenzyme overlay assay is a widely used tool for antibody-mediated identification of glycosphingolipids (GSLs) in mixtures. However, because the majority of GSLs is left unexamined in a chromatogram of a single assay, we developed a novel method that permits detection of various GSLs by sequential multiple immunostaining combined with individual coloring of GSLs in the same chromatogram. Specific staining was achieved by means of primary anti-GSL antibodies, directed against lactosylceramide, globotriaosylceramide, and globotetraosylceramide, in conjunction with alkaline phosphatase (AP)- or horseradish peroxidase (HRP)-conjugated secondary antibodies together with the appropriate chromogenic substrates. Triple coloring with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-AP, Fast Red-AP, and 3,3'-diaminobenzidine (DAB)-HRP resulted in blue, red, and black precipitates, respectively, following three sequential immunostaining rounds. Structures of antibody-detected GSLs were determined by direct coupling of TLC with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. This combinatorial technique was used to demonstrate structural GSL profiling of crude lipid extracts from human hepatocellular cancer. This powerful technology allows efficient structural characterization of GSLs in small tissue samples and marks a further step forward in the emerging field of glycosphingolipidomics.

Shiga toxin receptor Gb3Cer/CD77: tumor-association and promising therapeutic target in pancreas and colon cancer.

BACKGROUND: Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas. METHODOLOGY/PRINCIPAL FINDINGS: Tissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors. CONCLUSIONS/SIGNIFICANCE: Enhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.

Two-dimensional differential gel electrophoresis of a cell line derived from a breast cancer micrometastasis revealed a stem/ progenitor cell protein profile.

Dissemination of primary cancer cells to distant sites is an early event in breast cancer. These cells can invade the bone marrow, rest there, and many years later disseminated tumor cells (DTC) can grow out to form overt metastases. Epithelium specific cytokeratins are commonly used as marker proteins for sensitive detection of metastatic lesions. However, due to difficulties in the detection of DTC, the question arises if DTC necessarily have the same protein expression profile as advanced tumors. On that account, we analyzed the previously uncharacterized breast cancer DTC cell line BC-M1 by 2-D DIGE. Special protein concentration and purification protocols for 2-DE were developed which resulted in high recovery rates and increased display of alkaline proteins. A broad range reference map of metastasis relevant proteins was compiled including the cytokeratins 5, 7, 8, 17, 18, and 19 and several classes of cytoskeleton proteins involved in metastasis like ezrin, gelsolin, vinculin, or vimentin. BC-M1 shows the rare and highly metastatic vimentin/cytokeratin 5 positive and cytokeratin 8/18 negative breast cancer phenotype and expresses Her-2, which is also found in stem cells/progenitor cells of primary tumors. Supported by the detection of several other epithelium-derived proteins, the example BC-M1 indicates that the protein expression profile of DTC might be reminiscent of the expression profile of the early tumor, which differs from the advanced tumor. Hence, DTC from breast cancer patients' bone marrow expressed cytokeratin 5, which further supports our hypothesis.

The alpha2beta1 integrin-specific antagonist rhodocetin is a cruciform, heterotetrameric molecule.

The integrin alpha2beta1 plays an important role in various pathophysiological processes, such as thrombosis, wound healing, inflammation, and metastasis. Rhodocetin, a constituent of the venom of the hemorrhagic Malayan pit viper (Calloselasma rhodostoma), is a specific alpha2beta1 integrin antagonist. To understand its molecular mode of action, its structure was studied by crystallography. Its quaternary structure in solution was also analyzed biochemically. Two novel subunits of rhodocetin were sequenced by mass spectrometry. Their integrin binding was measured by protein interaction ELISAs. Rhodocetin is a C-type lectin-like protein (CLP) consisting of four homologous, yet distinct, subunits, alpha, beta, gamma, and delta, the latter two of which have been unknown to date. With their CLP folds and loop-swapping motifs, the subunits alpha, beta and gamma, delta form two heterodimeric pairs. Uniquely, they arrange orthogonally and shape a cruciform molecule. Bearing a single unpaired cysteine residue, rhodocetin can only form covalent supramolecular complexes with a maximum aggregation number of 2, unlike many heterodimeric CLPs. Being the first heterotetrameric CLP to be crystallized, rhodocetin provides not only the prototypic molecular structure for heterotetrameric CLPs, but also a lead structure for pharmaceutical alpha2beta1 integrin antagonists.

Structure analysis of N-glycoproteins.

General mass spectrometry-based strategies for analysis of N-glycosylated peptides are described. The well-established method utilizes Peptide-N-glycosidase F (PNGase F) for in-gel or in-solution release of N-linked glycans from the polypeptide chains (along with the conversion of the formerly N-glycosylated Asn to Asp), thus allowing separate analysis of glycan moieties and deglycosylated peptides. However, no assignment of individual glycans to a glycosylation site can be realized. Intact glycopeptides (i.e., proteolytic mixtures in which the glycan chains stay attached at their original glycosylation sites) can be analyzed either by a direct infusion or with HPLC separation prior to MALDI or ESI mass spectrometric analysis to provide both information on the glycan structure and glycosylation site in the same experiment. Several different strategies for efficient in-solution digestion of glycoproteins are described, such as proteolytic digestion in the electrospray capillary and simultaneous analysis of the resulting (glyco)peptides.

Tumor-associated CD75s- and iso-CD75s-gangliosides are potential targets for adjuvant therapy in pancreatic cancer.

Pancreatic adenocarcinoma confers one of the highest mortality rates in malignant human tumors with very poor prognosis. Because as yet no treatments are available that produce a substantial survival benefit for this fatal neoplasia, new therapeutic concepts are urgently required to support cancer standard treatment. In search of tumor-associated gangliosides with therapeutic background, we probed a random collection of cancerous and adjacent normal postoperative tissue samples from 38 patients for the expression of CD75s- and iso-CD75s-gangliosides. We exhaustively analyzed the expression of CD75s-1-ganglioside (IV(6)Neu5Ac-nLc4Cer) and structurally closely related iso-CD75s-1-ganglioside (IV(3)Neu5Ac-nLc4Cer) by means of immunohistology of cryosections and semiquantitative TLC of tissue lipid extracts combined with mass spectrometry. CD75s-1- and iso-CD75s-1-ganglioside showed an elevated expression in 42% and 66% of the tumors, respectively, indicating a significant association with neoplastic transformation (P = 0.001). Thus, increased expression of CD75s-1- and iso-CD75s-1-gangliosides renders these cell surface molecules promising candidates for oncologic applications. Further statistical analysis revealed a significant enhancement of CD75s-1-ganglioside in the group of less differentiated tumors (grade >2) suggesting this ganglioside as a potential marker for poor differentiation. The CD75s-specific antitumor drug rViscumin, which represents the recombinant counterpart of the ribosome-inactivating lectin viscumin, has successfully passed clinical phase I trials and provides an opportunity for treating pancreatic cancer. Consequently, if an enhanced expression is existent in malignant tissues, we propose the targeting of CD75s-gangliosides with rViscumin as a novel potential strategy in adjuvant treatment of pancreatic malignancies.

Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation.

Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.

Matching IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay and its clinical application for tracing tumor-associated glycosphingolipids in hepatocellular and pancreatic cancer.

Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including the development of cancer. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of modern mass spectrometry. Here we introduce direct coupling of IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay for the structural characterization of GSLs. We matched three complementary methods including (i) TLC separation of GSLs, (ii) their detection with oligosaccharide-specific proteins, and (iii) in situ MS analysis of protein-detected GSLs. The high specificity and sensitivity is demonstrated by use of antibodies, bacterial toxins, and a plant lectin. The procedure works on a nanogram scale, and detection limits of less than 1 ng at its best of immunostained GSLs were obtained. Furthermore, only crude lipid extracts of biological sources are required for TLC-IR-MALDI-MS, omitting any laborious GSL downstream purification procedures. This strategy was successfully applied to the identification of cancer-associated GSLs in human hepatocellular and pancreatic tumors. Thus, the in situ TLC-IR-MALDI-MS of immunolabeled GSLs opens new doors by delivering specific structural information of trace quantities of GSLs with only a limited investment in sample preparation.

Assessment of N-glycan heterogeneity of cactus glycoproteins by one-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Artificial environmental conditions in tissue culture, such as elevated relative humidity and rich nutrient medium, can influence and modify tissue growth and induce spontaneous changes from characteristic organization pattern to unorganized callus. As succulent plants with crassulacean acid metabolism, cacti are particularly susceptible to this altered growth environment. Glycosylated proteins of Mammillaria gracillis tissues cultivated in vitro, separated by SDS-PAGE, were detected with Con A after the transfer of proteins onto the nitrocellulose membrane. The glycan components were further characterized by affinity blotting with different lectins (GNA, DSA, PNA, and RCA(120)). The results revealed significant differences in glycoprotein pattern among the investigated cactus tissues (shoot, callus, hyperhydric regenerant, and tumor). To test whether the N-glycosylation of the same protein can vary in different developmental stages of cactus tissue, the N-glycans were analyzed by MALDI-TOF MS after in-gel deglycosylation of the excised 38-kDa protein band. Paucimannosidic-type N-glycans were detected in oligosaccharide mixtures from shoot and callus, while the hyperhydric regenerant and tumor shared glycans of complex type. The hybrid oligosaccharide structures were found only in tumor tissue. These results indicate that the adaptation of plant cells to artificial environment in tissue culture is reflected in N-glycosylation, and structures of N-linked glycans vary with different developmental stages of Mammillaria gracillis tissues.

Human gliosarcoma-associated ganglioside composition is complex and distinctive as evidenced by high-performance mass spectrometric determination and structural characterization.

Gangliosides (GGs), involved in malignant alteration and tumor progression/invasiveness, are considered as tumor biomarkers or therapeutic targets. Here, we describe the first systematic GG composition characterization in human gliosarcoma versus normal brain tissue using our recently developed mass spectrometry (MS) methods, based on nano-electrospray (nano-ESI), Fourier-transform ion cyclotron resonance (FT-ICR), and chip nano-ESI quadrupole time-of-flight (QTOF), complemented by thin-layer chromatographic (TLC) analysis and quantification. Combined MS enabled detection and structural assignment of 73 distinct GG species: many more than reported so far for investigated gliomas. Apart from the 7.4-times lower total GG content, gliosarcoma contained all major brain-associated species, however, in very altered proportions, exhibiting a highly distinctive pattern: GD3 (48.9%)>GD1a/nLD1>GD2/GT3>GM3>GT1b>GM2>GM1a/GM1b/nLM1>LM1>GD1b>GQ1b. MS also revealed abundant O-Ac-GD3; its sequencing provided structural evidence to postulate a novel O-Ac-GD3 isomer O-acetylated at the inner Neu5Ac-residue, previously not structurally confirmed. The high sensitivity and mass accuracy permitted the assignment of unusual minor species: GM4, Hex-HexNAc-nLM1, Gal-GD1, Fuc-GT1, GalNAc-GT1, O-Ac-GM3, di- O-Ac-GD3O-Ac-GD3, and O-Ac-GT3, not previously reported as glioma-associated. The gliosarcoma-expressed GA2 might represent a marker distinguishing astrocytic from oligodendroglial tumors. This is, to our knowledge, so far the most complete GG composition characterization of certain glioma, which demonstrates that our MS-based approach could provide essential structural information relevant to glycosphingolipid role(s) in brain tumor biology, differential diagnosis/prognosis and novel treatment concepts.

Mass spectrometry-based glycoproteomic approach involving lysine derivatization for structural characterization of recombinant human erythropoietin.

Lysine-containing peptides comprising glycosylation sites derived from recombinant human erythropoietin (rHuEPO) by trypsin or Lys-C and PNGase F dual digestion were derivatized with 2-methoxy-4,5-dihydro-1H-imidazole and its deuterated analogues. In the same reaction, under reducing conditions (beta-mercaptoethanol), cysteines were converted into methyl-cysteines and lysines into Lys-4,5-dihydro-1H-imidazole. Both modifications on cysteines and lysines simplified the CID-MS/MS spectra, while preserving the structural information by yielding y-series ions and improved the mass spectral signal intensity up to 25 times. Moreover, by this approach, the N-glycan occupation sites were unambiguously determined. O-Glycosylation sites as well as O-glycan structures were determined by a LC-MS/MS experiment carried out on dually digested rHuEPO. N-Glycan mixture purified on a graphitized carbon column using a newly developed method that extracted only sialylated carbohydrates was analyzed first using MALDI-TOF in negative linear ion mode with low mass accuracy but without interferences and metastabile ions and then a reflectron with high mass accuracy. After defining the precursor ions, we performed the nanoESI QTOF MS/MS analysis on N-glycans, mainly targeting the distinction between carbohydrates with sialylated antennae and those lacking sialic acid moieties.

Glycoproteomic survey of Mammillaria gracillis tissues grown in vitro.

The structure elucidation of protein-linked N-glycans in plants has raised interest in the past years due to remarkable physiological roles attributed to these modifications. However, little information about the glycoprotein patterns related to plant cell differentiation, dedifferentiation and transformation is available. In this work, the use of two-dimensional polyacrylamide gel electrophoresis in conjunction with matrix assisted laser/desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) for the characterization of carbohydrates released from plant glycoproteins is described. Proteins from different Mammillaria tissues (shoot, callus, hyperhydric regenerant, and TW tumor) were separated by 2D SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and incubated with Con A to detect N-glycosylated proteins. To discover if the same protein can have various N-glycan structures depending on the organization status of the tissue, the selected glycoprotein spot, which was common for all investigated tissues, was excised from the gels and digested by PNGase A. The released oligosaccharides were analyzed by MALDI-TOF-MS. The results obtained in this study indicate that the N-glycosylation pattern of the protein is clearly dependent on level of plant tissue organization and can be related to the specific morphogenic status.

Global Analysis of the Hortaea werneckii proteome: studying steroid response in yeast.

The response of the halophilic black yeast Hortaea werneckii to the steroid hormone progesterone has been studied at the protein level using fluorescent two-dimensional differential gel electrophoresis (2D-DIGE) technology in combination with mass spectrometry. Data on protein identification from this study reveal molecular mechanisms of the response to progesterone. In particular, the overexpression of Pck2 and Pac2 in the stimulated cells indicates the interactions of progesterone with the cell growth and reproduction signaling pathways.

Characterization of a molt-inhibiting hormone (MIH) of the crayfish, Orconectes limosus, by cDNA cloning and mass spectrometric analysis.

The structure of the precursor of a molt-inhibiting hormone (MIH) of the American crayfish, Orconectes limosus was determined by cloning of a cDNA based on RNA from the neurosecretory perikarya of the X-organ in the eyestalk ganglia. The open reading frame includes the complete precursor sequence, consisting of a signal peptide of 29, and the MIH sequence of 77 amino acids. In addition, the mature peptide was isolated by HPLC from the neurohemal sinus gland and analyzed by ESI-MS and MALDI-TOF-MS peptide mapping. This showed that the mature peptide (Mass 8664.29 Da) consists of only 75 amino acids, having Ala75-NH2 as C-terminus. Thus, C-terminal Arg77 of the precursor is removed during processing, and Gly76 serves as an amide donor. Sequence comparison confirms this peptide as a novel member of the large family, which includes crustacean hyperglycaemic hormone (CHH), MIH and gonad (vitellogenesis)-inhibiting hormone (GIH/VIH). The lack of a CPRP (CHH-precursor related peptide) in the hormone precursor, the size and specific sequence characteristics show that Orl MIH belongs to the MIH/GIH(VIH) subgroup of this larger family. Comparison with the MIH of Procambarus clarkii, the only other MIH that has thus far been identified in freshwater crayfish, shows extremely high sequence conservation. Both MIHs differ in only one amino acid residue ( approximately 99% identity), whereas the sequence identity to several other known MIHs is between 40 and 46%.

Tumor-associated CD75s gangliosides and CD75s-bearing glycoproteins with Neu5Acalpha2-6Galbeta1-4GlcNAc-residues are receptors for the anticancer drug rViscumin.

The anticancer drug rViscumin, currently under clinical development, has been shown in previous studies to be a sialic acid specific ribosome inactivating protein (RIP). Comparative binding assays with the CD75s-specific monoclonal antibodies HB6 and J3-89 revealed rViscumin to be a CD75s-specific RIP due to identical binding characteristics toward CD75s gangliosides. The receptor gangliosides are IV6nLc4Cer, VI6nLc6Cer, and the newly characterized ganglioside VIII6nLc8Cer, all three carrying the Neu5Acalpha2-6Galbeta1-4GlcNAc motif. To elucidate the clinical potential of the rViscumin targets, CD75s gangliosides were determined in several randomly collected gastrointestinal tumors. The majority of the tumors showed an enhanced expression of CD75s gangliosides compared with the unaffected tissues. The rViscumin binding specificity was further investigated with reference glycoproteins carrying sialylated and desialylated type II N-glycans. Comparative Western blots of rViscumin and ricin, an rViscumin homologous but galactoside-specific RIP, revealed specific recognition of type II N-glycans with CD75s determinants by rViscumin, whereas ricin failed to react with terminally sialylated oligosaccharides such as CD75s motifs and others. This strict binding specificity of rViscumin and the increased expression of CD75s gangliosides in various tumors suggest this anticancer drug as a promising candidate for an individualised adjuvant therapy of human tumors.