Jining Liu

A comparison of fetal and adult porcine islets with regard to Gal alpha(1,3)Gal expression and the role of human immunoglobulins and complementin islet cell cytotoxicity.

BACKGROUND It is still debated whether fetal or adult porcine islets should be the preferred choice for future clinical islet xenotransplantation. Each type of islet preparation has advantages and disadvantages compared with the other. Here we present a direct comparison between fetal and adult porcine islets with regard to Gal alpha(1,3)Gal expression, immunoglobulin and complement binding, and cytotoxicity after exposure to fresh human serum. METHOD Islet single cell suspensions were prepared from adult and fetal islets by trypsin digestion. Fluorescein isothiocyanate-conjugated Bandeiraea simplicifolia isolectin B4 (BS-IB4) and affinity-purified chicken anti-Gal alpha(1,3)Gal antibody was used to detect Gal alpha(1,3)Gal expression. Immunoglobulin and complement binding to the islet cells and cytotoxicity for islet cells was compared after incubation with fresh and heat-inactivated human sera and with an immune serum from a diabetic patient who received a fetal porcine islet transplant. Furthermore, two pools of human AB sera were depleted of porcine endothelial cell cytotoxic human anti-Gal alpha(1,3)Gal antibodies by absorption and were used to analyze the effect of Gal alpha(1,3)Gal antibody removal on islet cell cytotoxicity. RESULTS Fetal islet cells readily bound both BS-IB4 and the chicken anti-Gal alpha(1,3)Gal antibody. None of 10 adult porcine islet preparations were stained by BS-IB4. In comparison, IgY anti-Gal Ab binding was detected in two of eight adult islet isolations, whereas the other six preparations showed marginal/no binding. After incubation of fetal islet cells with fresh human serum, C3c binding was strongly positive and IgM binding variable, with occasional binding of IgG and no detectable binding of IgA. Adult islet cells were also strongly positive for C3c but did not bind detectable amounts of IgM, IgG, or IgA. Immune sera from a patient who had received fetal porcine islets showed the presence of induced antibodies that bound to fetal islet cells and to porcine peripheral blood lymphocytes, whereas binding to adult islet cells was barely detectable. Fresh human sera showed a high and similar level of complement-mediated lytic activity for both adult islet cells (78+/-22%) and fetal islet cells (75+/-16%). Cytotoxicity for fetal islet cells and peripheral blood lymphocytes was significantly reduced when the corresponding sera were depleted of anti-Gal antibodies before use (P=0.002 and P=0.003, respectively). In contrast, no difference in cytotoxicity for adult islet cells was detected when anti-Gal-depleted human sera were used. CONCLUSION Gal alpha(1,3)Gal expression is occasionally detectable on adult porcine islet cells, but not as readily and at a lower level, compared with fetal islet cells. Thus, as porcine fetal islets mature to adult islets, the expression of the Gal alpha(1,3)Gal epitope gradually diminishes. Consequently, cytotoxic anti-Gal alpha(1,3)Gal antibodies in human serum play an important role in the lysis of fetal but not adult porcine islet cells.

Removal of xenoreactive human anti-pig antibodies by absorption on recombinant mucin-containing glycoproteins carrying the Gal alpha1,3Gal epitope.

BACKGROUND The hyperacute rejection caused by preformed natural antibodies in the recipient species reacting with donor species endothelial antigens is one of the major obstacles preventing routine use of clinical xenotransplantation. Based on the known structure and biosynthetic pathway of the major porcine xenoantigen, Gal alpha1,3Gal, we developed a novel strategy aimed at specific removal of human, natural anti-pig antibodies. METHODS Cotransfection of COS cells with expression plasmids encoding a secreted mucin/immunoglobulin chimera and the porcine alpha1,3-galactosyltransferase facilitated simple immunoaffinity purification of a highly Gal alpha1,3Gal-substituted mucin/immunoglobulin fusion protein from transfected cell supernatants. RESULTS Cotransfection of COS cells resulted in a mucin/Ig concentration in the supernatants ranging from 150 to 200 ng/ml. Approximately 300 ng of mucin/Ig chimeras absorbed onto 50 microl of packed anti-mouse IgG agarose beads could completely remove cytotoxic human anti-pig antibodies from 1 ml of human AB serum, as estimated in porcine endothelial cell cytotoxicity assays. Purified human IgG, IgM, and IgA all bound porcine endothelium, but only IgG and IgM were cytotoxic in the presence of rabbit complement. When the cytotoxicity of human IgG at 8 mg/ml and IgM at 1 mg/ml was completely removed by absorption on the mucin/Ig chimera, the binding to porcine endothelium was only partly reduced. Antibodies mediating antibody-dependent cellular cytotoxicity against porcine endothelium were also absorbed, indicating the importance of Gal alpha1,3Gal epitopes for this effect. CONCLUSIONS We describe the construction and production of a new and effective Gal alpha1,3Gal-substituted, mucin domain-containing absorber that can be used in a pretransplant extracorporeal immunoabsorption setting to remove anti-pig antibodies involved in antibody-dependent, complement- and cell-mediated cytotoxicity of pig endothelial cells.

Mucin-type fusion proteins with blood group A or B determinants on defined O-glycan core chains produced in glycoengineered Chinese hamster ovary cells and their use as immunoaffinity matrices

Assays for quantification, and methods for removal, of anti-A and anti-B antibodies are the key for the success of ABO incompatible organ transplantation programs. In order to produce tools that can be used as substrates in tests for anti-A/anti-B quantification and specificity determination or as affinity matrices in extracorporeal immunoadsorption (IA) columns, we engineered Chinese hamster ovary (CHO) cells secreting mucin-type fusion proteins carrying blood group A or B determinants on defined O-glycan core saccharide chains. Besides the P-selectin glycoprotein ligand-1/mouse immunoglobulin G(2b) (PSGL-1/mIgG(2b)) cDNA, CHO cells were transfected with plasmids encoding core 2 (β1,6GlcNAc-T1) or core 3 (β1,3GlcNAc-T6 and β1,3Gal-T5) enzymes together with α1,2Fuc-T1 or α1,2Fuc-T2 and the A or B gene-encoded α1,3GalNAcT or α1,3Gal-T, respectively. Selected clones with the correct glycophenotype were expanded and cultured in shaker flasks and Wave bioreactors. Western blotting was used to characterize purified fusion protein and liquid chromatography-mass spectrometry was used to characterize the released O-glycans. Clones producing PSGL-1/mIgG(2b) carrying O-glycans with A and B determinants on type 1 (Galβ3GlcNAc), type 2 (Galβ4GlcNAc) and type 3 (Galβ3GalNAcα) outer core saccharide chains were established. The conversion of CHO cells from exclusive inner core 1 (Galβ3GalNAc) to core 3 (GlcNAcβ3GalNAc) O-glycan producers was almost complete, whereas conversion to inner core 2 (GlcNAcβ6GalNAc) O-glycans was incomplete as was the α2-fucosylation of the core 1 chain. Sialylation may prevent these biosynthetic steps. The clinical utility of the blood group A and B substituted mucin-type fusion proteins as substrates in enzyme-linked immunosorbent assay or as affinity matrices in IA columns is explored.

O-glycan repertoires on a mucin-type reporter protein expressed in CHO cell pools transiently transfected with O-glycan core enzyme cDNAs

Glyco-engineering of host cells is used to increase efficacy, decrease immunogenicity and increase circulatory half-lives of protein biopharmaceuticals. The effect of transiently expressed O-glycan core chain glycosyltransferases on O-glycan biosynthesis pathways in CHO cells is reported. Liquid chromatography-mass spectrometry and Western blotting were used to map the O-glycome of a mucin-type fusion protein transiently co-transfected with β1,3-N-acetylglucosaminyltransferase 3 (extended C1 β3GnT3), core 2 β1,6-N-acetylglucosaminyltransferase I (C2 β3GnT1) or core 3 β1,3-N-acetylglucosaminyltransferase 6 (C3 β3GnT6) in CHO cells. Extended core 1 (GlcNAcβ1,3Galβ1,3GalNAc) and core 3 (GlcNAcβ1,3GalNAc), and increased expression of core 2 [Galβ1,3(GlcNAcβ1,6)GalNAc], O-glycans were generated on P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL1/mIgG2b). Endogenous poly-N-acetyllactosamine (poly-LacNAc) synthase elongated extended core 1 and core 3 generating O-glycans with up to five LacNAc repeats. Low amounts of core 3 O-glycans appeared upon extended C1 β3GnT3 expression. The α2,6-sialylated type 2 chain was detected upon co-transfection with the β-galactoside α2,6-sialyltransferase I. N-acetylglucosamine-6-O-sulfotransferase 2 transferred sulfate to carbon 6 of GlcNAc in poly-LacNAc sequences. CHO cells with its known O-glycan repertoire can be used to express recombinant mucin-type proteins together with selected glycosyltransferases in order to recreate carbohydrate determinants on defined O-glycan chains. They will become important tools for assessing the core chain-dependent binding activity of carbohydrate-binding proteins.

Avian influenza H5 hemagglutinin binds with high avidity to sialic acid on different O-linked core structures on mucin-type fusion proteins.

The interaction between P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b) fusion protein carrying multiple copies of the influenza hemagglutinin receptor Siaα2-3Gal on different O-glycan chains and recombinant human influenza H5N1 A/Vietnam/1203/04 hemagglutinin was investigated with a Biacore biosensor. The fusion protein was produced by stable cell lines in large scale cultures and purified with affinity- and gel filtration chromatography. The C-P55 and 293-P cell lines were established by transfecting the Chinese hamster ovary (CHO)-K1 and Human embryonic kidney (HEK)-293 cell lines with plasmids encoding the PSGL-1/mIgG2b fusion protein, while the C-PSLex cell line was engineered by transfecting CHO-K1 cells with the plasmids encoding the core 2 β1,6GnT-I and FUT-VII glycosyltransferases. Glycosylation was characterized by lectin Western blotting of the proteins and liquid chromatography - mass spectrometry of released non-derivatized O-glycans. Biacore experiments revealed that PSGL-1/mIgG2b is a good binding partner of H5. The binding curves displayed a slow dissociation indicating a multivalent binding. The H5 hemagglutinin binds with similar strength to PSGL-1/mIgG2b carrying mostly sialylated core 1 (clone C-P55), a mix of sialylated core 1 and sialylated lactosamine (clone 293-P) or mainly sialylated lactosamine (clone C-PSLex) O-glycans, indicating that this hemagglutinin is unable to discriminate between these structures. The potential use of the large, flexible PSGL-1/mIgG2b mucin-type fusion protein carrying Siaα2-3Gal as a multivalent inhibitor of influenza virus is discussed.

Mucin-type proteins produced in the Trichoplusia ni and Spodoptera frugiperda insect cell lines carry novel O-glycans with phosphocholine and sulfate substitutions

The O-glycans of a recombinant mucin-type protein expressed in insect cell lines derived from Trichoplusia ni (Hi-5) and Spodoptera frugiperda (Sf9) were characterized. The P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b) fusion protein carrying 106 potential O-glycosylation sites and 6 potential N-glycosylation sites was expressed and purified from the Hi-5 and Sf9 cell culture medium using affinity chromatography and gel filtration. Liquid chromatography mass spectrometry (LC-MS) of O-glycans released from PSGL-1/mIgG2b revealed a large repertoire of structurally diverse glycans, which is in contrast to previous reports of only simple glycans. O-Glycans containing hexuronic acid (HexA, here glucuronic acid and galacturonic acid) were found to be prevalent. Also sulfate (Hi-5 and Sf9) and phosphocholine (PC; Sf9) O-glycan substitutions were detected. Western blotting confirmed the presence of O-linked PC on PSGL-1/mIG2b produced in Sf9 cells. To our knowledge, this is the first structural characterization of PC-substituted O-glycans in any species. The MS analyses revealed that Sf9 oligosaccharides consisted of short oligosaccharides (<6 residues) low in hexose (Hex) and with terminating N-acetylhexosamine (HexNAc) units, whereas Hi-5 produced a family of large O-glycans with (HexNAc-HexA-Hex) repeats and sulfate substitution on terminal residues. In both cell lines, the core N-acetylgalactosamine was preferentially non-branched, but small amounts of O-glycan cores with single fucose or hexose branches were found.

Adsorption of chain type–specific ABO antibodies on Sepharose‐linked A and B tetrasaccharides

BACKGROUND: Antigen‐specific removal of anti‐A and anti‐B on immunoadsorption columns carrying the blood group A and B trisaccharides is one important component of some protocols used in ABO‐incompatible organ transplantation. Because ABO antibodies exist requiring parts of the core saccharide chain for binding, the anti‐A and ‐B–binding capacity of individual and combined, Sepharose‐linked Types 1 through 4 A and B tetrasaccharides with that of the A and B trisaccharides was compared.

Shiga-like toxin binds with high avidity to multivalent O-linked blood group P1 determinants on mucin-type fusion proteins

The binding of Shiga-like toxin 1 (Stx1) and Shiga-like toxin 2 (Stx2) to a mucin-like fusion protein, P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-1/mIgG2b), carrying multiple copies of the blood group P1 determinant on O-glycans was investigated with western blot and the biosensor Biacore. Chinese hamster ovary K-1 (CHO-K1) cells were stably transfected with linearized plasmids encoding the PSGL-1/mIgG2b fusion protein, the pigeon α1,4-galactosyltransferase (α4Gal-T) and the core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT-I). Western blot analyses of purified PSGL-1/mIgG2b and liquid chromatography-mass spectrometry (LC-MS) of released O-glycans confirmed the presence of the P1 determinant. Western blot analysis indicated strong binding of Stx1, but not Stx2, to PSGL-1/mIgG2b. In a Biacore assay, Stx1 and Stx2 were immobilized on a dextran chip and the binding of purified PSGL-1/mIgG2b and a P(k)-albumin neoglycoprotein was analyzed. Stx1 and Stx2 bound with high avidity to both PSGL-1/mIgG2b and P(k)-albumin, while the Stx1 binding was the strongest. In summary, we have shown that the pigeon α4Gal-T can be aberrantly expressed in CHO cells together with the core 2 enzyme to generate multiple, O-linked P1 determinants on a simultaneously expressed mucin-type fusion protein. P1-decorated PSGL-1/mIgG2b bound with high avidity to both Stx1 and Stx2, and as such constitutes a potential therapeutic inhibitor of these toxins.

A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins

Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO) cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b), CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1), core 3 (B3GNT6), core 4 (GCNT1 and B3GNT6), or extended core 1 (B3GNT3) chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galβ3GlcNAc) or type 2 (Galβ4GlcNAc) outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins.

Engineering of Therapeutic and Diagnostic O -Glycans on Recombinant Mucin-Type Immunoglobulin Fusion Proteins Expressed in CHO Cells

Metabolic engineering of mammalian cells for optimized glycosylation is usually done to improve activity and the pharmacokinetic features of glycoprotein therapeutics. The field is mainly focused around engineering of N-glycans. We have created a platform in which recombinant mucin-type immunoglobulin fusion proteins are used as scaffolds for multivalent expression of O-glycans with diagnostic or therapeutic potential. The methods used to make stable CHO cell lines secreting a mucin-type fusion protein with blood group A or B determinants following expression of up to five different cDNAs are described.