Electrophoretic studies of the cystic fibrosis ciliary inhibitor and its interaction with immunoglobulin G

The cystic fibrosis ciliary inhibitor (CFCI) has been partially purified from serum and plasma of cystic fibrosis (CF) homozygotes and heterozygotes, and from media of cultured fibroblasts derived from cystic fibrosis genotypes. Characterization and comparison of fractions containing the CFCI were carried out by polyacrylamide gel electrophoresis. Gel electrophoresis confirmed previous molecular weight estimations of 4,500 to 11,000 for the CFCI and provided an estimate of the number of proteins present in the fractions. Low molecular weight proteins from serum and media were combined with IgG preparations. No specific binding to IgG by the media fraction containing the CFCI could be demonstrated by the techniques employed. There was decreased binding of the low molecular weight serum fraction containing CFCI to native IgG molecules from cystic fibrosis patients as compared to IgG from normal individuals. However, IgG from CF individuals demonstrated increased binding of the cfci-containing low molecular weight serum fraction after gel filtration in the presence of guanidinium chloride. This suggests: 1) that very low concentrations of CFCI are present in media fractions; and 2) that native CF IgG cannot bind the low molecular weight CFCI fractions to the same degree as native IgG from normals or CF IgG that has been dissociated from non-covalently bound components.     

Localization of the binding site for transforming growth factor-beta in human alpha2-macroglobulin to a 20-kDa peptide that also contains the bait region

alpha2-Macroglobulin (alpha2M) functions as a major carrier of transforming growth factor-beta (TGF-beta) in vivo. The goal of this investigation was to characterize the TGF-beta-binding site in alpha2M. Human alpha2M, which was reduced and denatured to generate 180-kDa subunits, bound TGF-beta1, TGF-beta2, and NGF-beta in ligand blotting experiments. Cytokine binding was not detected with bovine serum albumin that had been reduced and alkylated, and only minimal binding was detected with purified murinoglobulin. To localize the TGF-beta-binding site in alpha2M, five cDNA fragments, collectively encoding amino acids 122-1302, were expressed as glutathione S-transferase (GST) fusion proteins. In ligand blotting experiments, TGF-beta2 bound only to the fusion protein (FP3) that includes amino acids 614-797. FP3 bound 125I-TGF-beta1 and 125I-TGF-beta2 in solution, preventing the binding of these growth factors to immobilized alpha2M-methylamine (alpha2M-MA). The IC50 values were 33 +/- 5 and 26 +/- 6 nM for TGF-beta1 and TGF-beta2, respectively; these values were comparable with or lower than those determined with native alpha2M or alpha2M-MA. A GST fusion protein that includes amino acids 798-1082 of alpha2M (FP4) and purified GST did not inhibit the binding of TGF-beta to immobilized alpha2M-MA. FP3 (0.2 microM) neutralized the activity of TGF-beta1 and TGF-beta2 in fetal bovine heart endothelial (FBHE) cell proliferation assays; FP4 was inactive in this assay. FP3 also increased NO synthesis by RAW 264.7 cells, mimicking an alpha2M activity that has been attributed to the neutralization of endogenously synthesized TGF-beta. Thus, we have isolated a peptide corresponding to 13% of the alpha2M sequence that binds TGF-beta and neutralizes the activity of TGF-beta in two separate biological assays.     

Purification and partial amino acid sequences of the binding protein from Bombyx mori for CryIAa delta-endotoxin of Bacillus thuringiensis

The binding protein for Bacillus thuringiensis delta-endotoxin, CryIAa, from the brush border membrane of the midgut of Bombyx mori was purified by the dot blot method and delta-endotoxin affinity chromatography. The binding protein was purified to 235-fold enrichment from cholic acid extracts of brush border membranes from B. mori midgut by activated CryIAa-affinity chromatography and DEAE ion-exchange chromatography. The purified binding protein showed a single band of 180 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and this band specifically reacted to 125I-labeled CryIAa on Immobilon membrane. The affinity of the binding protein for CryIAa was equivalent to that of the brush border membrane vesicles and solubilized membrane proteins. Partial amino acid sequences of the binding protein showed sequence similarity to the cadherin-like binding protein for CryIAb from Manduca sexta, but not for CryIAc binding protein from M. sexta and Heliothis virescens.     

Alpha 2-macroglobulin of rabbits inhibits the habutobin activity

We have investigated whether alpha 2-macroglobulin (alpha 2M) of rabbits inhibits the activity of habutobin, a thrombin-like enzyme from Trimeresurus flavoviridis venom. Rabbit alpha 2M was purified with ultracentrifugation, gel filtration on Sepharose 6B and ion exchange chromatography on DEAE-Sephacel. Inhibitory effects of rabbit alpha 2M on habutobin was determined by the fibrin forming activity, digestion of A alpha chain of fibrinogen, and the release of fibrinopeptide A from fibrinogen. As a results, purified alpha 2M showed a single band with high molecular weight, around 800,000 mol. wt by means of polyacrylamide gel electrophoresis using PhastSystem. Besides inhibiting amidolytic and caseinolytic activity of porcine trypsin, it has inhibited the activity of habutobin: that is, in the presence of rabbit alpha 2M, fibrin forming activity of habutobin was decreased and habutobin-induced digestion of A alpha chain was inhibited. In addition, rabbit alpha 2M reduced habutobin-induced release of fibrinopeptide A from rabbit fibrinogen.     

The binding of phytohemagglutinin M to rat spleen lymphocytes. Quantitative studies

Phytohemagglutinin M (PHAM) has been purified from the commercial mixture of proteins produced by Phaseolus vulgaris, using a Sepharose-thyroglobulin column. The protein gave one band on gel electrophoresis and two bands on SDS-gel electrophoresis (mol. wt. 33 700 and 32 100, respectively). Molecular weight determination by ultracentrifugation gave a value of 61 200 +/- 700. The protein had a minimum sugar content of 16%. Binding studies of PHAM to purified rat spleen lymphocytes have been performed at 0, 25, and 37 degrees C. It was shown that the cells bound about the same amount of lectin at 0 and 37 degrees C, but less protein was bound at 25 degrees C. The binding phenomenon showed saturability at all temperatures. Data were analyzed by Scatchard plots and two kinds of binding sites were found. High-affinity sites and low-affinity sites have been characterized in terms of association constants and (apparent) number. It was also shown that cells treated with trypsin or sodium azide bound less lectin. Bound concanavalin A did not appear to affect the amount of bound PHAM, but its influence was reflected in the value of the association constant for the binding of PHAM. Unlabelled PHAM was shown to displace radioactive PHAM from the cells, but could not remove bound concanavalin A. The significance of these results is discussed in terms of the fluid plasma membrane model and cellular metabolism.     

Hypersensitivity to chironomid larvae

BACKGROUND: The aim of this study was to identify the proteolytic activity which triggers the transformation of human alpha2-macroglobulin (alpha2-M) in seminal fluid and its binding to its receptor. METHODS: Measurement of the concentrations of total and transformed alpha2-M in seminal fluid was accomplished by ELISA. Zymography of seminal plasma was performed in SDS-polyacrylamide gels containing casein as proteolytic substrate. Rate electrophoresis, SDS-PAGE, and Western blotting were applied to study the complex formation of prostate-specific antigen (PSA) with alpha-M. Ligand-binding analysis of sperm cells was performed using [125I] labeled proteins. Detection of receptor on sperm cells was achieved by immunofluorescence. RESULTS: The mean concentration of total alpha2-M in a random collection of seminal plasma was 4.6 microg/ml. On average, between 33-98% of the inhibitor was found to be transformed. Zymography of seminal plasma revealed a proteolytic activity which is associated with a 33-kDa protein identified as PSA. Its proteolytic activity could be inhibited by 0.2-M. Both purified PSA and seminal plasma were capable of transforming native alpha2-M. Binding of PSA to alpha2-M triggers the exposition of receptor binding sites in the inhibitor molecule, which causes binding of the complex to alpha2-M-R/LRP identified on spermatozoa. CONCLUSIONS: PSA, the main proteinase in seminal fluid, is responsible for the transformation of alpha2-M and for its binding to alpha2-M-R/LRP present on spermatozoa. The binding of alpha2-M-PSA complexes to the spermatozoa receptor may exert an impact on normal sperm-cell functions.     

Purification and characterization of GDP-L-Fuc: N-acetyl beta-D-glucosaminide alpha1-->6fucosyltransferase from human blood platelets

c-6-L-Fucosyltransferase (alpha1,6FucT; EC 2.4.1.68) from human platelets, the enzyme that is released into serum during coagulation of blood, was purified 100,000-fold. The purification required three sequential chromatographic steps: chromatofocusing, affinity column chromatography on GnGn-Gp(asialo-aglacto-transferrin glycopeptide)-CH-Sepharose, and gel filtration of Sephadex G-200. The final preparation contained a protein that migrated as a single discrete band Mr of 58,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, and as a single enzymatically active peak Mr of 58,000 in gel filtration. Although the purified enzyme utilized the biantennary GnGn-Gp as substrate, it was twice as active with the triantennary oligosaccharide when the Man alpha1,3 antenna was substituted with GlcNacbeta1,4. On the other hand the tetraantennary oligosaccharide was not a preferred substrate. The Km values for the substrate asialo-agalactotransferrin-glycopeptide, and GDP-L-fucose were 29 and 28 microM, respectively. The optimum pH of the enzyme was 6.0. The activity of alpha1,6FucT was abolished in the presence of beta-mercaptoethanol. Divalent cations such as Mg2+ and Ca2+ activated, but Cu2+, Zn2+ and Ni2+ strongly inhibited the activity.     

Purification and characterization of a nuclear DNA-binding phosphoprotein in fetal and tumor tissues

The basic nonhistone phosphoprotein 110/8.4 (M.W. X 10(-3)/pI) was found in 0.35 M NaCl nuclear extracts of four tumor tissues, i.e., fast-growing Novikoff hepatoma, Morris hepatoma 3924A, HeLa cells, and Namalwa cells; it was also found in fetal rat liver. This protein was not detected in normal or regenerating liver and thus may represent an "oncofetal" protein of potential interest as a cancer "marker." Protein 110/8.4 was purified approximately 4000- to 5000-fold under nondenaturing condition from 0.35 M NaCl nuclear extracts of Novikoff hepatoma cells or Namalwa cells by ammonium sulfate fractionation, calcium phosphate gel treatment, and phosphocellulose chromatography. Sodium dodecyl sulfate:polyacrylamide gel electrophoresis of the purified native protein revealed a single polypeptide chain with a molecular weight of approximately 110,000. The pI of the protein was estimated to be 8.4 by nonequilibrium pH gradient electrophoresis in 9 M urea; accordingly, this protein was designated 110/8.4. Amino acid analysis showed that Protein 110/8.4 had an acidic:basic amino acid ratio of 1.25 and a high lysine and serine content; approximately 20% of the serine residues were found to be phosphorylated. Hydrazinolysis indicated that the carboxyl-terminal amino acid was serine; the amino terminus appeared to be blocked. Binding of Protein 110/8.4 to DNA was studied by the nitrocellulose filter assay. High-affinity binding occurred at ionic strength equal to or below 0.15 M.     

Alpha2-macroglobulin associates with beta-amyloid peptide and prevents fibril formation

We have used the yeast two-hybrid system to isolate cDNAs encoding proteins that specifically interact with the 42-aa beta-amyloid peptide (Abeta), a major constituent of senile plaques in Alzheimer's disease. The carboxy terminus of alpha2-macroglobulin (alpha2M), a proteinase inhibitor released in response to inflammatory stimuli, was identified as a strong and specific interactor of Abeta, utilizing this system. Direct evidence for this interaction was obtained by co-immunoprecipitation of alpha2M with Abeta from the yeast cell, and by formation of SDS-resistant Abeta complexes in polyacrylamide gels by using synthetic Abeta and purified alpha2M. The association of Abeta with alpha2M and various purified amyloid binding proteins was assessed by employing a method measuring protein-protein interactions in liquid phase. The dissociation constant by this technique for the alpha2M-Abeta association using labeled purified proteins was measured (Kd = 350 nM). Electron microscopy showed that a 1:8 ratio of alpha2M to Abeta prevented fibril formation in solution; the same ratio to Abeta of another acute phase protein, alpha1-antichymotrypsin, was not active in preventing fibril formation in vitro. These results were corroborated by data obtained from an in vitro aggregation assay employing Thioflavine T. The interaction of alpha2M with Abeta suggests new pathway(s) for the clearance of the soluble amyloid peptide.     

Some characteristics of cytosol binding protein for 1alpha,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol in rat intestinal mucosa

Cytosol binding protein for 1alpha,25-(OH)2D3 and 25-OHD3 was demonstrated in rat intestinal mucosa using sucrose density gradient ultracentrifugation analysis, Sephadex G-200 column chromatography and polyacrylamide disc gel electrophoresis. The binding protein has a sedimentation constant of 5-6S, a molecular weight of 100,000-120,000 daltons and a mobility in electrophoresis of Rf 0.42. Further analysis of binding behavior by DEAE-cellulose filter assays revealed that the bindig protein had a higher affinity for 25-OHD3 than for 1alpha,25-(OH)2D3, and that 1alpha,25-(OH)2D3 competed with 25-OHD3 for the same binding site on the protein. The apparent dissociation constant for 1alpha,25-(OH)2D3 and 25-OHD3 was 9.2 X 10(-9) M and 1.5 X 10(-9) M, respectively. The binding capacity was 3.1 pmoles/mg protein for both 1alpha,25-(OH)2D3 and 25-OHD3. The order of binding affinity was 25-OHD3 greater than 1alpha,25-(OH)2D3 greater than D3 = 1alpha-OHD3.     

Alpha7 and alpha8 nicotinic receptor subtypes immunopurified from chick retina have different immunological, pharmacological and functional properties

Nicotinic receptors are present in the chick retina, but their structure and functional characteristics are still unclear. Using anti-alpha7 and anti-alpha8 subunit-specific antibodies, we immunopurified the alpha7 and alpha8 subtypes of chick retina neuronal nicotinic receptors. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the two purified subtypes consistently showed a similar peptide composition characterized by the presence of two major peptides of M(r) 58 +/- 1 and 54 +/- 1 kDa, and two minor peptides of 67 and 61 +/- 1 kDa. In the alpha7 subtype, the 58 kDa peptide was recognized by anti-alpha7 but not by anti-alpha8 antibodies; in the alpha8 subtype, the 58 kDa peptide was recognized only by anti-alpha8 antibodies. The alpha7 subtype had a single class of [125I]alpha-bungarotoxin binding sites with a K(D) value of 1.2 nM, whereas the purified alpha8 subtype had two classes of binding sites, one with a K(D) of 5.5 nM and the other with very high affinity (KD 52 pM), but present in only 8% of the receptors. Competition binding experiments also showed the presence on the alpha8 subtype of high- and low-affinity classes of binding sites; the affinity for cholinergic drugs of the former was greater than that of the single class present on the alpha7 subtype. When reconstituted in planar lipid bilayers, both subtypes formed ligand-gated cation channels with major conductance levels of 42 and 52 pS but with different lifetimes; the two channels were activated by agonists and blocked by d-tubocurarine and the glycinergic antagonist strychnine. In line with the binding data, the reconstituted alpha8 subtype had greater agonist sensitivity than the alpha7 subtype.     

Association of apolipoprotein E with alpha2-macroglobulin in human plasma

Apolipoprotein (apo) E plays a central role in the transport of lipids among different organs and cell types, whereas alpha2-macroglobulin (alpha2M) is responsible for the binding and inactivation of plasma proteases, as well as the transport of various cytokines, growth factors, and hormones. In the present study, evidence is presented for direct binding of apoE with alpha2M in human plasma, based on the observation that two-dimensional non-denaturing gradient gel electrophoretic separation of plasma resulted in co-migration of apoE with alpha2M in a complex intermediate in size (18.5 nm in diameter) between low (LDL) and high density lipoproteins (HDL). ApoE associated with alpha2M could be immunoprecipitated from plasma with anti-human alpha2M antiserum. Purified apoE, labeled with 125I, bound to native and methylamine-activated alpha2M (alpha2M-MA) in vitro in a time- and concentration-dependent manner. ApoE bound to alpha2M-MA with greater affinity than alpha2M. The binding of apoE to both alpha2M and alpha2M-MA did not depend on the presence of lipid. Ingestion of an oral fat load resulted in a reduction in the amount of apoE associated with alpha2M. Sphingomyelin vesicles and very low density lipoproteins (VLDL), but not phosphatidylcholine vesicles or HDL3, inhibited the in vitro binding of 125I-labeled apoE3 to alpha2M and alpha2M-MA. Binding of 125I-labeled apoE3 was also partially inhibited by an excess of platelet-derived growth factor and beta-amyloid protein, but not interferon-gamma. Subjects with an apoE 4/4 phenotype had less apoE associated with alpha2M in plasma than subjects with an apoE 3/3 or 2/2 phenotype, corresponding to reduced in vitro binding of apoE4 with alpha2M or alpha2M-MA. Although the functional significance of apoE binding to alpha2M remains to be determined, the present results demonstrate that: 1) apoE is non-covalently bound to alpha2M in human plasma, 2) alpha2M-MA has a greater capacity to bind apoE than alpha2M, 3) various proteins or lipoproteins known to bind apoE or alpha2M can potentially affect the interaction of apoE with alpha2M, and 4) association of apoE with alpha2M or alpha2M-MA is dependent on apoE phenotype.     

Restoration of 125I-alpha-bungarotoxin binding activity to the alpha subunit of Torpedo acetylcholine receptor isolated by gel electrophoresis in sodium dodecyl sulfate

The four subunits (alpha, beta, gamma, delta) of the acetylcholine receptor from Torpedo californica have been isolated by preparative gel electrophoresis in sodium dodecyl sulfate. After removal of the sodium dodecyl sulfate by dialysis of the polypeptides against a cholate-containing buffer, the alpha subunit, but not the other chains, binds 125I-alpha-bungarotoxin in a saturable manner. The binding affinity, 0.1-0.2 microM, is approximately 10(4)-fold lower than that observed for native acetylcholine receptor. For three preparations of alpha subunit, 1 mol of subunit bound 0.87, 0.38, and 0.33 mol of 125I-alpha-bungarotoxin at saturation. The binding was inhibited by cholinergic ligands, although the apparent affinities of these ligands for alpha were 50-100-fold lower than that found for the native receptor. These results indicate that at least part of the alpha-bungarotoxin binding site resides on the alpha subunit.     

Isolation and characterization of rat olfactory marker protein

The olfactory marker protein was isolated and characterized from rat olfactory bulbs. Its properties and those of the olfactory marker protein isolated from the mouse are described. The rat protein was less acidic (pI = 5.0) than the mouse protein (pI = 4.7). However, the amino acid compositions were very similar: in both proteins arginine plus lysine accounted for 13 mol% and glutamate plus aspartate for 30 mol% of the total residues. Molecular weights of both proteins estimated by sodium dodecyl sulfate gel electrophoresis were indistinguishable and estimated to be 16,500. The molecular weight of the native rat olfactory marker protein estimated by gel filtration techniques was 30,000, which is identical to the molecular weight of the native mouse and garfish olfactory marker proteins. This suggested a dimeric structure. The purified rat and mouse proteins behaved like species of 35,000 molecular weight on gel filtration.     

Left ventricular diastolic function in young adults: the Coronary Artery Risk Development in Young Adults Study

Shiga-like toxin type I (SLT-I) is a cytotoxin produced by certain strains of Escherichia coli. SLT-I is a bipartite molecule comprised of A (SLT-IA) and B (SLT-IB) subunits. In holotoxin, five B subunits are arranged in a pentameric ring and bind to globotriaosylceramide receptors on the surface of susceptible eukaryotic cells. The SLT-IB pentamer is noncovalently associated with one A subunit that has N-glycosidase activity and ultimately causes the death of targeted cells. Using a previously described overexpression vector, plasmid SBC32, we developed a two-step procedure for the purification of biologically active recombinant SLT-IB. Periplasmic proteins were extracted from E. coli JM105(pSBC32), fractionated by ammonium sulfate precipitation, and separated by isoelectric focusing in a pH 3-10 gradient. SLT-IB was present in fractions with pH values between 5.0 and 6.0, consistent with its reported pI of 5.8. SLT-IB was purified to homogeneity in a second step by native polyacrylamide gel electrophoresis. Purified SLT-IB was characterized for biological and biochemical activity. When analyzed by native polyacrylamide gel electrophoresis, the majority of SLT-IB had an apparent molecular weight of 38,900, consistent with a pentameric subunit association. Chemical cross-linking of SLT-IB with disuccinimidyl suberate resulted in species with molecular weights corresponding to dimeric, trimeric, tetrameric, and pentameric forms of B subunit. SLT-IB was not cytotoxic to Vero cells at concentrations as high as 10 micrograms/ml and protected Vero cells from native SLT-I. Purified SLT-IB maintained its ability to associate with SLT-IA to form holotoxin that exhibited toxicity similar to native toxin.     

Functional domains of Escherichia coli single-stranded DNA binding protein as assessed by analyses of the deletion mutants

A series of C- and N-terminal deletion mutants of Escherichia coli single-stranded DNA binding protein (SSB) was constructed, purified, and characterized in terms of ability to self-multimerize and to bind to DNA. High-performance gel filtration chromatography revealed that the amino acids 89-105 play a key role in the maintenance of homotetramer for native SSB of 177 amino acids. Interestingly, all of the N-terminal deletion mutants studied here were eluted as octamers, indicating that the N-terminal 11 residues are involved in the prevention of the formation of octamers. The binding of SSB and its deletion mutant proteins to single-stranded d(T)n was examined by gel mobility shift assay and circular dichroism spectroscopy. C-terminal deletion mutant proteins, SSB1-135 and SSB1-115, maintained high affinity and may be wrapped by single-stranded DNA (ssDNA) in the same way as in the case of native SSB. In contrast, deletion of the C-terminal region (residues 89-115) or N-terminal region (residues 1-11) caused a dramatic decrease in the binding affinity. Furthermore, two different stoichiometries of SSB in the complexes with d(T)64, but not with d(T)32, were observed for native SSB, SSB1-135, SSB1-115, and SSB37-177, suggesting that the (SSB)65 and (SSB)35 binding modes, as previously demonstrated [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-7802], occurred at lower and higher SSB concentrations, respectively. A functional map for SSB molecule was presented and discussed.     

Prostate specific antigen (PSA) value adjusted for transition zone volume and free PSA (gamma-seminoprotein)/PSA ratio in the diagnosis of prostate cancer in patients with intermediate PSA levels

OBJECTIVE: To identify binding proteins of leptin in human plasma. METHODS: Binding was evaluated by electrophoresis, size exclusion chromatography (SEC), Western blotting, and radioisotope labeling. Quantification of leptin and the different forms of alpha2-macroglobulin (alpha2-M) was performed by ELISA. RESULTS: Leptin interacts with the proteinase inhibitor, alpha2-M. 125I-labeled leptin specifically binds to the transformed inhibitor, which arises by reaction with proteinases or with reactive primary amines. No leptin binding was observed to the native alpha2-M, which abundantly occurs in plasma. The complex formation between leptin and alpha2-M was found to proceed within minutes and was stable, as it resisted separation by SEC and electrophoresis. The Kd of the complex was 2.14 +/- 0.78 micromol/l. Complex formation with transformed alpha2-M did not interfere with the immunological determination of leptin in plasma. The leptin-alpha2-M complex was found to be recognized by the alpha2-M receptor/low density lipoprotein receptor-related protein. By computer analysis, a simple model is presented showing that the degree of transformation of alpha2-M may significantly influence the leptin concentration in blood. CONCLUSIONS: The proteinase inhibitor, alpha2-M, may act as a leptin-binding protein in human plasma. Binding of leptin to transformed alpha2-M and its rapid clearance by the alpha2-M receptor may significantly influence the bioavailability of leptin in human plasma.     

An improved method for the purification of rat serum albumin: removal of contaminants by concanavalin A-Sepharose

Minor contaminants occasionally found in conventionally prepared rat serum albumin were easily and completely removed by concanavalin A-Sepharose chromatography. The unadsorbed fraction from a concanavalin A-Sepharose column contained albumin which was homogeneous on polyacrylamide gel electrophoresis. The recovery of albumin form rat serum was approximately 30%. Approximately 2% of the added protein obtained as an albumin peak in DEAE-cellulose chromatography was adsorbed on and eluted with alpha-methyl-D-glucoside from the concanavalin A-Sepharose column, and resolved into three components by gel electrophoresis. There was one major glycoprotein, possibly alpha 1-antitrypsin, and two minor proteins one of which was albumin.     

Comparative study of target antigens for primate xenoreactive natural antibodies in pig and rat endothelial cells

BACKGROUND: A rat-to-primate cardiac xenograft model has been proposed as an alternative to the clinically relevant but more cumbersome pig-to-primate model for assessing the efficacy of strategies aimed at preventing xenograft hyperacute rejection. As in pig xenografts, the rejection of rat hearts was mediated by the binding of xenoreactive natural antibodies (XNA) and complement activation. The present study was conducted to identify target antigens recognized by cynomolgus and rhesus monkey IgM XNA on rat tissues and cells in comparison with pig cells. METHODS: The reactivity of rhesus or cynomolgus serum on pig and rat endothelial cells (ECs) was studied by flow cytometry, ELISA, and complement-dependent cytotoxicity, after removal of primate XNA by perfusion of pig livers, immunoadsorption on a Gal alpha(1,3)Gal affinity column, and enzymatic removal of alpha-galactosyl epitopes from the cell surface. Rat and pig EC extracts were also immunoprecipitated with primate serum and resolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The expression of the Gal alpha(1,3)Gal epitope was analyzed on rat tissues and ECs by immunohistochemistry, flow cytometry, and Western blot, using the isolectin B4 from Griffonia simplicifolia. RESULTS: Removal of primate XNA or of alphaGal epitopes resulted in a decrease in XNA binding to pig and rat cells, leaving a similar degree of residual reactivity in the two species. At least five proteins of 260, 210, 110, 56, and 50 kDa were immunoprecipitated on rat ECs, with molecular weight similar to several proteins identified on pig ECs. These results suggest that primate XNA recognize similar antigens on rat and pig ECs. Rat cells expressed lower levels of the Gal alpha(1,3)Gal epitope than pig cells. A large proportion, but not all, of primate XNA react with this epitope on pig and rat ECs. CONCLUSION: This study suggests that the rat is a valuable species for the evaluation of genetic engineering strategies on the vascular endothelium aimed at preventing hyperacute xenograft rejection.