The identification of cell surface antigens is critical to the development of new diagnostic and therapeutic modalities for the management of prostate cancer. Prostate stem cell antigen (PSCA) is a prostate-specific gene with 30% homology to stem cell antigen 2, a member of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA encodes a 123-aa protein with an amino-terminal signal sequence, a carboxyl-terminal GPI-anchoring sequence, and multiple N-glycosylation sites. PSCA mRNA expression is prostate-specific in normal male tissues and is highly up-regulated in both androgen-dependent and -independent prostate cancer xenografts. In situ mRNA analysis localizes PSCA expression in normal prostate to the basal cell epithelium, the putative stem cell compartment of the prostate. There is moderate to strong PSCA expression in 111 of 126 (88%) prostate cancer specimens examined by in situ analysis, including high-grade prostatic intraepithelial neoplasia and androgen-dependent and androgen-independent tumors. Flow cytometric analysis demonstrates that PSCA is expressed predominantly on the cell surface and is anchored by a GPI linkage. Fluorescent in situ hybridization analysis localizes the PSCA gene to chromosome 8q24.2, a region of allelic gain in more than 80% of prostate cancers. A mouse homologue with 70% amino acid identity and similar genomic organization to human PSCA has also been identified. These results support PSCA as a target for prostate cancer diagnosis and therapy.

https://www.pnas.org/content/pnas/95/4/1735.full.pdf

Prostate stem cell antigen: A cell surface marker overexpressed in prostate cancer

This review describes the application of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to carbohydrate analysis and covers the period 1991-1998. The technique is particularly valuable for carbohydrates because it enables underivatised, as well as derivatised compounds to be examined. The various MALDI matrices that have been used for carbohydrate analysis are described, and the use of derivatization for improving mass spectral detection limits is also discussed. Methods for sample preparation and for extracting carbohydrates from biological media prior to mass spectrometric analysis are compared with emphasis on highly sensitive mass spectrometric methods. Quantitative aspects of MALDI are covered with respect to the relationship between signal strength and both mass and compound structure. The value of mass measurements by MALDI to provide a carbohydrate composition is stressed, together with the ability of the technique to provide fragmentation spectra. The use of in-source and post-source decay and collision-induced fragmentation in this context is described with emphasis on ions that provide information on the linkage and branching patterns of carbohydrates. The use of MALDI mass spectrometry, linked with exoglycosidase sequencing, is described for N-linked glycans derived from glycoproteins, and methods for the analysis of O-linked glycans are also covered. The review ends with a description of various applications of the technique to carbohydrates found as constituents of glycoproteins, bacterial glycolipids, sphingolipids, and glycolipid anchors.

Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates.

Tissue trauma or invasion by pathogens or parasites induce changes in the quantities of several macromolecules in animal body fluids These changes comprise one aspect of the acute phase response (APR), which in toto involves metabolic changes in several organ systems One clear indication of the response is the increase in synthesis and secretion by the liver of several plasma proteins, with simultaneous decreases in others These acute phase proteins (APP) function in a variety of defense-related activities such as limiting the dispersal of infectious agents, repair of tissue damage, inactivation of proteases, killing of microbes and other potential pathogens, and restoration of the healthy state Some APP are directly harmful to microbes, while others modify targets thus marking them for cell responses Some work alone while others contribute to cascades Proteins that are APP in mammals, and that have been identified in both teleosts and elasmobranchs include C-reactive protein, serum amyloid P, and several components of the Complement system Others reported in teleosts include transferrin and thrombin Of these, only CRP has been reported to increase in acute phase plasma In trout, a precerebellin-like protein is an APP with unknown functions A cDNA library enriched in fragments of transcripts that were more abundant in livers from fish undergoing an APR recently yielded sequences resembling 12 additional known APP, and as many others either not known to be APP, or not similar to others yet in public databases It appears that, as in mammals, hepatocytes are the prime source of APP in fish, and that pro-inflammatory cytokines induce transcription of their genes

The acute phase response and innate immunity of fish

Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors.

Prions are the causative agent of the transmissible spongiform encephalopathies, such as Creutzfeldt-Jakob disease in humans. In these prion diseases the normal cellular form of the prion protein (PrP(C)) undergoes a post-translational conformational conversion to the infectious form (PrP(Sc)). PrP(C) associates with cholesterol- and glycosphingolipid-rich lipid rafts through association of its glycosyl-phosphatidylinositol (GPI) anchor with saturated raft lipids and through interaction of its N-terminal region with an as yet unidentified raft associated molecule. PrP(C) resides in detergent-resistant domains that have different lipid and protein compositions to the domains occupied by another GPI-anchored protein, Thy-1. In some cells PrP(C) may endocytose through caveolae, but in neuronal cells, upon copper binding to the N-terminal octapeptide repeats, the protein translocates out of rafts into detergent-soluble regions of the plasma membrane prior to endocytosis through clathrin-coated pits. The current data suggest that the polybasic region at its N-terminus is required to engage PrP(C) with a transmembrane adaptor protein which in turn links with the clathrin endocytic machinery. PrP(C) associates in rafts with a variety of signalling molecules, including caveolin-1 and Fyn and Src tyrosine kinases. The clustering of PrP(C) triggers a range of signal transduction processes, including the recruitment of the neural cell adhesion molecule to rafts which in turn promotes neurite outgrowth. Lipid rafts appear to be involved in the conformational conversion of PrP(C) to PrP(Sc), possibly by providing a favourable environment for this process to occur and enabling disease progression.

https://www.tandfonline.com/doi/pdf/10.1080/09687860500449994

The prion protein and lipid rafts.

CD59 (protectin) is a glycophosphoinositol (GPI)-anchored inhibitor of the membrane attack complex of complement found on blood cells, endothelia and epithelial cells. In addition to the lipid-tailed CD59, soluble lipid-free forms of CD59 are present in human body fluids. We have investigated the detailed structural composition of the naturally occurring soluble urinary CD59 (CD59u) using peptide mapping, anion-exchange chromatography, sequential exoglycosidase digestion and matrix-assisted laser-desorption mass spectrometry (MALDI-MS). CD59u exhibited an average M(r) of 12444 in MALDI-MS. Mass analysis of the isolated C-terminal peptide (T9) indicated that a GPI-anchor (at Asn-77) without an inositol-associated phospholipid was present in soluble CD59u. By using residue-specific exoglycosidases, chemical modification and MALDI-MS structures of seven different GPI-anchor variants were determined. Variant forms of the anchor had deletions and/or extensions of one or more monosaccharide units. Sialic acid linked to an N-acetylhexosamine-galactose arm was found in two GPI-anchor variants. The N-linked carbohydrate side chain of CD59u (at Asn-18) also displayed considerable heterogeneity. The predominant oligosaccharide chains were fucosylated biantennary and triantennary complexes with variable sialylation. Mono Q anion-exchange chromatography resolved urinary CD59 into nine different fractions that bound equally well to the terminal complement SC5b-8 complexes. Despite binding to C5b-8, soluble CD59u inhibited complement lysis at an approx. 200-fold lower efficiency than erythrocyte CD59. These results document the structural heterogeneity of both the GPI anchor and N-linked oligosaccharide of CD59 and demonstrate that the phospholipid tail is needed for the full functional activity of CD59. The site of cleavage between the diradylglycerol phosphate and inositol suggests that a mammalian phospholipase D could be involved in the solubilization of GPI-anchored proteins.

Structural composition and functional characterization of soluble CD59: heterogeneity of the oligosaccharide and glycophosphoinositol (GPI) anchor revealed by laser-desorption mass spectrometric analysis.

https://www.kidney-international.org/article/S0085-2538(15)58961-8/pdf

Urinary excretion of protectin (CD59), complement SC5b-9 and cytokines in membranous glomerulonephritis

Activation of the complement system has been documented in both experimental and clinical studies of acute myocardial infarction (AMI). Our earlier immunohistochemical studies have shown that the deposition of the membrane attack complex (MAC) of complement is associated with the loss of protectin (CD59), a glycosyl-phosphatidylinositol (GPI)-anchored sarcolemmal regulator of MAC, from the human and rat infarcted myocardium. In this study we detected, using an enzyme immunoassay (EIA), CD59 in the plasma of AMI patients at a concentration of 23.0 ± 8.4 ng/ml (mean ± SD; n = 17) at 4 h and 27.3 ± 11.8 ng/ml (n = 24) at 24 h after AMI. Both values were significantly higher than in healthy controls (7.8 ± 6.4 ng/ml; n = 20; P < 0.001). The amount of CD59 correlated with the level of soluble terminal complement complexes (SC5b-9; r = 0.84; P < 0.01) in the plasmas of AMI patients. Our results suggest that myocardial damage leads to release of CD59 from the sarcolemmal cell membranes during AMI.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-3083.2000.00783.x

Detection of a Soluble Form of the Complement Membrane Attack Complex Inhibitor CD59 in Plasma after Acute Myocardial Infarction

https://www.kidney-international.org/article/S0085-2538(15)58889-3/pdf

Activation of the terminal complement cascade in renal infarction.

Idiopathic membranous glomerulonephritis (iMGN) has previously been shown to be associated with urinary excretion of terminal complement complexes while increased urinary levels of cytokines have been reported in mesangial proliferative glomerulonephritis. In the present cross-sectional study urinary excretion of IL-1 beta, TNF-alpha, IL-6, and soluble C5b-9 (SC5b-9) was examined for 23 patients with iMGN, 16 patients with diabetic nephropathy (DNP), and 17 healthy subjects. IL-1 beta excretion (pg/mg crea) was significantly higher in iMGN patients (375, range 162-11,000) than in DNP patients (39, range 22-59, P < 0.001) or healthy controls (151, range 23-481, P < 0.001). TNF-alpha excretion rate (pg/mg crea) was clearly higher (38, range 21-700) in iMGN patients than in DNP patients (14, range 8-52, P < 0.001) or healthy subjects (11, range 7-26, P < 0.001). Median IL-6 excretion (pg/mg crea) was only marginally higher in iMGN patients (73, range 0-850) than in healthy subjects (64, range 3-158, P = 0.02) but significantly higher than in DNP patients (29, range 17-47, P < 0.001). No significant correlation with corresponding serum values was observed for urinary IL-6 or TNF-alpha excretion. Urinary IL-1 beta and TNF-alpha correlated with decreased renal function. Five of 23 patients showed progression of iMGN over a follow-up of 6 months. The excretion of all cytokines, TNF-alpha in particular, was significantly higher in patients with a progressive disease than in the other patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Timo Lehto

Papers

Structural composition and functional characterization of soluble CD59: heterogeneity of the oligosaccharide and glycophosphoinositol (GPI) anchor revealed by laser-desorption mass spectrometric analysis.

Urinary excretion of protectin (CD59), complement SC5b-9 and cytokines in membranous glomerulonephritis

Detection of a Soluble Form of the Complement Membrane Attack Complex Inhibitor CD59 in Plasma after Acute Myocardial Infarction

Activation of the terminal complement cascade in renal infarction.

Urinary excretion of cytokines and complement SC5b-9 in idiopathic membranous glomerulonephritis