Identification of precursor forms of free prostate-specific antigen in serum of prostate cancer patients by immunosorption and mass spectrometry.

The structural features of the free prostate-specific antigen (F-PSA) present in human blood have not been clarified up to now, and it is, therefore, not known why F-PSA is not complexed by the protease inhibitors that are present in human blood in large amounts. This lack of information is mainly attributable to the low amount of F-PSA in serum, which makes the isolation and structural characterization very difficult, especially when only limited amounts of individual sera are available. It has now been demonstrated that F-PSA occurs as a mixture of different pro-PSA forms (zymogen forms) in the sera of prostate cancer patients, and that, in some of these sera, a form with the regular NH2 terminus of PSA is present as well. Among the five serum samples investigated, all contained the (-7), (-5), and (-4) pro-PSA forms, whereas the (-1) and (-2) forms were only present in three of them. These three samples also contained the form with the regular NH2 terminus. The (-3) and (-6) pro-PSA forms have not been detected thus far. The F-PSA has been isolated by immunosorption from the individual sera using streptavidin-coated magnetic beads. The pro-PSA forms were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry after producing peptides by endoproteinase from Lysobacter enzymogenes digestion of the SDS-PAGE-separated F-PSA band. The structural identity of the (-7)pro-PSA form was further proven by sequencing of that particular peptide using electrospray ionization quadrupole time-of-flight mass spectrometry.

Analysis of free prostate-specific antigen (PSA) after chemical release from the complex with alpha(1)-antichymotrypsin (PSA-ACT).

BACKGROUND: Prostate-specific antigen (PSA), a marker for prostate cancer (CaP), forms a covalent complex with alpha(1)-antichymotrypsin (ACT) in human blood. Structural analysis of the PSA-ACT complex is difficult, and complexation may be a reason for biased immunological assays when compared with the analysis of free PSA. We developed a method to cleave the PSA-ACT complex chemically. The liberated PSA was thus available for analysis as free PSA (F-PSA). METHODS: PSA was released from the PSA-ACT complex by cleaving the interprotein ester bond with ethanolamine under alkaline conditions. The release was followed by reversed-phase HPLC and an immunoassay for F-PSA. Released PSA obtained from human blood was further immunopurified and analyzed by matrix-assisted laser desorption-induced time of flight (MALDI-TOF) mass spectrometry. RESULTS: In vitro-prepared PSA-ACT complex was completely cleaved by treatment with nucleophilic compounds such as ethanolamine at pH 9-10. The released PSA was stable under these conditions and could be measured by reversed-phase HPLC as well as the ENZYMUN immunoassay for F-PSA. When plasma from a CaP patient [containing 190 microg/L F-PSA and 1890 microg/L total PSA (T-PSA)] was treated under similar conditions, a concentration of approximately 1600 microg/L F-PSA was measured at the end of the incubation, indicating that the PSA-ACT complex was completely cleaved. Two benign prostatic hyperplasia and CaP sera panels (12 and 13 sera, respectively) containing 4-45 microg/L T-PSA were similarly treated. The concentrations of F-PSA measured after incubation were, on average, 85% of the T-PSA values of the untreated sera. Finally, the PSA released from the complex of the CaP plasma was isolated by immunosorption, analyzed by MALDI-TOF mass spectrometry, and compared to PSA obtained from semen. The intact PSA as well as the peptides observed after digestion with endoproteinase Lys C did not reveal any structural difference between the PSA from these two sources. CONCLUSIONS: PSA complexed to ACT in plasma of a CaP patient seems to be structurally very similar to the PSA reference material from semen. The release of PSA from the PSA-ACT complex allows F-PSA and T-PSA to be measured by the same immunological assay, thus eliminating any possible bias between two different assays.

Purification of prostate-specific antigen from human serum by indirect immunosorption and elution with a hapten.

When isolating proteins from complex biological material by immunosorption, the nonspecific binding and elution of other proteins present in much larger concentrations than the target protein are often a general problem, preventing the isolation of a pure protein. To improve this situation we have developed a new indirect immunosorption method, which makes use of a digoxigenylated anti-analyte antibody. This antibody is linked to streptavidin-coated magnetic beads via a biotinylated anti-digoxigenin antibody. After binding of the analyte to the affinity matrix, the complex composed of analyte and digoxigenylated anti-analyte antibody is specifically eluted with a solution of digoxigenin-lysine at pH 7.3. Coelution of nonspecifically bound proteins was highly reduced as revealed by SDS-PAGE when compared to the acidic eluates of the direct immunosorption. The efficiency of the indirect immunosorption method was demonstrated with the isolation of free prostate-specific antigen (PSA) and PSA/alpha(1)-antichymotrypsin complex from human serum and subsequent analysis of the intact proteins by SDS-PAGE and MALDI-TOF-mass spectrometry.

Beyond plant lectin histochemistry: preparation and application of markers to visualize the cellular capacity for protein-carbohydrate recognition.

Oligosaccharides can store biological information. In this respect, their capacity even outmatches that of oligo- and polymeric structures of nucleotides and amino acids. Protein-carbohydrate interactions are thus considered to be involved in the regulation of diverse cellular activities. Over decades, plant lectins have proven valuable for assessing structural aspects of the enormous variety of carbohydrate epitopes and for monitoring spatially and/or temporally restricted patterns of expression. If the presence of these epitopes and the alterations in their occurrence bear physiological relevance, one reasonable possibility is that the visualized saccharides serve as ligands in an operative protein-carbohydrate recognition system. To support the validity of this hypothesis, receptor sites for a sugar compound must be localized. Carrier-immobilized carbohydrates (neoglycoconjugates) are adequate for this purpose. Chemical synthesis gains access to such probes. In the first stage, the presence of binding sites such as lectins in the tissue is ascertained. The next step toward proving the outlined hypothesis is the application of the first localized then purified endogenous receptors as glycohistochemical markers. It is essential to point out that the fine specificities of plant and animal lectins can differ, although they share an identical monosaccharide specificity. Thus, neoglycoconjugates for localizing sugar ligand-binding proteins and endogenous lectins to detect suitable binding partners are promising probes to enhance our knowledge about the capacities of cells to be engaged in protein-carbohydrate recognition in situ.

Identification of carbohydrate deficient transferrin forms by MALDI-TOF mass spectrometry and lectin ELISABiochim Biophys Acta 1998 Aug 24;1381(3):356.

Transferrin was isolated from sera of patients with severe alcohol abuse and from control sera by affinity chromatography using an immobilized polyclonal antibody from sheep, followed by gel filtration. The purified transferrin was then separated by MonoQ chromatography. Compared to the controls, sera from heavy alcohol consumers showed two additional transferrin peaks, eluting earlier than the three main transferrin forms present in all sera. Further analysis of the isolated transferrin forms by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and enzyme linked immunosorbent assay with different digoxigenylated lectins (lectin ELISA) revealed that the main carbohydrate deficient transferrin (CDT) forms are lacking either one or both of the N-Glycan chains.

Neoglycoproteins with the synthetic complex biantennary nonasaccharide or its alpha 2,3/alpha 2,6-sialylated derivatives: their preparation, assessment of their ligand properties for purified lectins, for tumor cells in vitro, and in tissue sections, and their biodistribution in tumor-bearing mice.

Neoglycoproteins were prepared with chemoenzymatically synthesized complex biantennary N-glycan derivatives the nonreducing ends of which bear typical sequences found in glycoproteins. A chemically obtained biantennary heptasaccharide-azide was reduced and acylated with a 6-aminohexanoyl spacer. Elongation of the deprotected heptasaccharide using glycosyltransferases yielded a biantennary nonasaccharide with terminal galactose residues and two undecasaccharides terminating with alpha 2,6- or alpha 2,3-linked sialic acid. The free amino group of the spacer of these oligosaccharides was converted into an isothiocyanate. Its subsequent coupling to bovine serum albumin gave neoglycoproteins with a yield of 2.4-3.6 glycan chains per carrier molecule. This versatile synthetic pathway allows employment of a wide variety of complex-type glycans, which can be introduced to various test systems in vitro and in vivo to evaluate potential biomedical applications. Solid-phase assays with biotinylated sugar receptors revealed discriminatory binding properties of the three neoglycoproteins, especially for the mistletoe lectin. This direct assay system is preferable to the measurement of inhibitory capacities with respect to model ligands. Ligand type- and cell type-dependent quantitative differences in the binding properties of the probes were detected by FACScan analyses with a panel of tumor cell lines and by monitoring of staining in tissue sections for small cell and non-small-cell lung cancer and mesotheliomas. Biodistribution of iodinated neoglycoproteins in mice gave a prolonged presence of the sialylated probes in serum. Relative to the nonasaccharide, the uptake, especially of the iodinated neoglycoprotein with alpha 2,3-sialylated ligand chains, was clearly elevated in mice for kidneys and Ehrlich tumors. On the basis of the documented feasibility of these applications, it is concluded that the further elaboration of glycan chain variants by the described synthetic approach in combination with the given test panel is warranted to evaluate the potential of complex glycan chain-carrying neoglycoproteins for diagnostic and therapeutic purposes.

Chemoenzymatic synthesis of a sialylated diantennary N-glycan linked to asparagine.

A partial structure of many glycoproteins, a glycosylated asparagine carrying a complex type undecasaccharide N-glycan (Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man alpha 1-3) [Neu5Ac(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4) GlcNAc(beta 1-4)GlcNAc-Asn) was obtained by total synthesis. As a starting material served a chemically synthesized diantennary heptasaccharide azide which was deprotected in a three-step sequence in high yield. The reduction of the anomeric azide was accomplished with propanedithiol in methanol-ethyldiisopropylamine. Coupling of the glycosyl amine to an activated aspartic acid gave the benzyl protected asparagine conjugate. After removal of the six benzyl functions the resulting free heptasaccharide asparagine was elongated enzymatically in the oligosaccharide part. The use of beta-1,4-galactosyltransferase and alpha-2,6-sialytransferase in the presence of alkaline phosphatase allowed the efficient transfer of four sugar units to the acceptor resulting in a full length N-glycan, a sialyated diantennary undecasaccharide-asparagine of the complex type.

Synthesis of glycopeptides and neoglycoproteins containing the fucosylated linkage region of N-glycoproteins.

N-Glycoproteins fucosylated in the core region occur in tumor membranes and virus envelopes. Partial structures of such N-glycoproteins containing fucosylated chitobiosyl asparagine conjugates were synthesized using the allyloxycarbonyl (Aloc) and the tert-butyl ester protecting groups in the peptide portion. As the alpha-fucosidic bond of the conjugates revealed to be very sensitive to acids when carrying ether-type protecting groups, a method for exchanging the protecting groups of the fucose portion of saccharides was developed. Conjugates containing O-acetyl protected fucose proved to be stable against acids used in glycopeptide syntheses. These methods were applied in the synthesis of a fucosyl chitobiose hexapeptide with the partial sequence of a leukemia virus envelope glycoprotein. The glycopeptide was coupled to bovine serum albumin yielding a neoglycoprotein which contains a glycoconjugate of exactly specified structure.

Chemical and enzymatic synthesis of multivalent sialoglycopeptides.

Linear and branched glycopeptides containing multiple sialyl-N-acetyllactosamine side chains have been synthesized using a combined chemical and enzymatic approach. Peptide backbones in which beta-GlcNAc-Asn residues were incorporated were obtained in good yields by optimized solid-phase synthesis following the Boc strategy. The resulting multivalent glycopeptides were galactosylated in near-quantitative yields using bovine galactosyltransferase, UDP-galactose, and calf alkaline phosphatase that destroys the inhibiting side product UDP. Subsequent enzymatic sialylation yielded the desired glycopeptides containing asparagine-linked sialyl-N-acetyllactosamine side chains. The compounds were characterized by 1H NMR and FABMS. Recombinant sialyltransferase and CMP-sialate synthetase were used for the enzymatic synthesis of sialosides on a preparative scale. The synthetic glycopeptides were tested as inhibitors of influenza virus to cells, revealing that most of the multivalent sialoglycopeptides exhibit increased binding that depends on the spacing when compared to monovalent compounds. A possible mechanism for increased binding is proposed.

Synthetic tumor-associated glycopeptide antigens.

Glycopeptides with TN antigen (GalNAc)Ser/Thr and T-antigen structures (beta Gall-3GalNAc)Ser/Thr, described as tumor-associated antigens, were synthesized and coupled to bovine serum albumin. Alternatively, synthetic methods for the construction of beta-anomeric analogues of the TN and T-antigen glycopeptides were developed, aiming at antigenic structures having a varied stereochemistry of the linkage between the carbohydrate and the peptide moiety. As a further type of potential tumor-associated antigen, fucosyl-chitobiose asparagine glycopeptides were synthesized, deprotected, and coupled to bovine serum albumin. The chemical methods developed now make the complex sensitive glycoprotein partial structures accessible in analytically pure form and in preparative amounts.