Lonely killers: Effector cell- and complement-independent non-proapoptotic cytotoxic antibodies inducing membrane lesions

The majority of the most effective monoclonal antibodies (mAbs) currently in the clinics bind to cancer or immune cells. Classic mechanisms of cell killing by therapeutic mAbs include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and induction of apoptosis by engagement of specific cell ligands. A few reports have described mAbs whose cytotoxic activity is Fc-independent and that do not induce the morphological and biochemical changes associated with the apoptosis-type of cell death. Even fewer works describe mAbs able to directly induce membrane lesions. Here, we discuss the available data on those molecules and their cell killing activity, with particular attention to the case of a mAb specific for the tumor-associated N-glycolyl (Neu5Gc)-GM3 ganglioside [GM3(Neu5Gc)]. Some similarities are found in the cell death pathways triggered by these mAbs, but data are not abundant. We conclude that the usefulness of mAbs with a direct cytotoxic activity for immunotherapeutic strategies deserves deeper research.Key words: cytotoxicity, therapeutic antibody, necrosis, membrane lesion, cell death     

Effects of natural human antibodies against a nonhuman sialic acid that metabolically incorporates into activated and malignant immune cells

Humans are genetically incapable of producing the mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), due to an inactivating mutation in the enzyme synthesizing it. Despite this, human cells and tissues appear capable of metabolically incorporating Neu5Gc from exogenous sources, including dietary red meat and dairy products. All normal humans studied are now shown to have circulating Abs against Neu5Gc, with marked differences in isotype levels. The question arises whether such Abs can adversely affect Neu5Gc-expressing human cells or tissues. In this study, we show that although normal human PBMC do not incorporate Neu5Gc during in vitro incubation, activated T cells do. Primary human leukemia cells and human leukemic cell lines are even more efficient at incorporation. Human sera containing naturally high levels of anti-Neu5Gc IgG Abs (hereafter abbreviated GcIg) deposited complement on Neu5Gc-expressing leukemic cells and activated T cells, but not on normal cells. The binding of GcIg resulted in complement-mediated cytotoxicity, which was inhibited by heat inactivation. Low anti-Neu5Gc IgG-containing human sera did not mediate any of these effects. Mixed killing assays confirmed the 15-fold selective killing of leukemic cells over PBMC by GcIg following Neu5Gc feeding. This approach could potentially serve as novel way to target malignant cells for death in vivo using either natural Abs or anti-Neu5Gc Abs prepared for this purpose. Further studies are needed to determine whether deposition of natural GcIg and complement can also target healthy proliferating immune cells for death in vivo following incorporation of dietary Neu5Gc.     

A shift from N-glycolyl- to N-acetyl-sialic acid in the GM3 ganglioside impairs tumor development in mouse lymphocytic leukemia cells

Humans, in contrast to other mammals, do not synthesize N-glycolyl-neuraminic acid (Neu5Gc) due to a deletion in the gene (cmah) encoding the enzyme responsible for this conversion, the cytidine monophospho-N-acetyl-neuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase). The detection of considerable amounts of Neu5Gc-sialoconjugates, in particular gangliosides, in human malignancies makes these antigens attractive targets for immunotherapy, in particular with monoclonal antibodies (mAbs). We have previously described a GM3(Neu5Gc) ganglioside-specific mAb, named 14F7, with the ability to kill tumor cells in a complement-independent manner. Silencing the cmah gene in GM3(Neu5Gc)-expressing L1210 mouse lymphocytic leukemia B cells caused the abrogation of this cytotoxic effect. We now show that cmah-silenced L1210 cells (cmah-kd) express a high level of GM3(Neu5Ac) and have an impaired ability for anchorage-independent cell growth and tumor development in vivo. No evidences of increased immunogenicity of the cmah-kd cell line were found. These results provide new evidences on the role of GM3(Neu5Gc), or Neu5Gc-sialoconjugates in general, in tumor biology. As an important tool in this study, we used the humanized version (here referred to as 7C1 mAb) of a recently described, rationally-designed mutant of 14F7 mAb that is able to bind to both GM3(Neu5Gc) and GM3(Neu5Ac). In contrast to its parental antibody, the humanized 14F7 (14F7hT) mAb, 7C1 mAb was able to kill not only GM3(Neu5Gc)-expressing L1210 wild type cells, but also GM3(Neu5Ac)-expressing cmah-kd cells, which endorses this antibody as a potential agent for cancer immunotherapy.     

Human embryonic stem cells express an immunogenic nonhuman sialic acid

Human embryonic stem cells (HESC) can potentially generate every body cell type, making them excellent candidates for cell- and tissue-replacement therapies. HESC are typically cultured with animal-derived 'serum replacements' on mouse feeder layers. Both of these are sources of the nonhuman sialic acid Neu5Gc, against which many humans have circulating antibodies. Both HESC and derived embryoid bodies metabolically incorporate substantial amounts of Neu5Gc under standard conditions. Exposure to human sera with antibodies specific for Neu5Gc resulted in binding of immunoglobulin and deposition of complement, which would lead to cell killing in vivo. Levels of Neu5Gc on HESC and embryoid bodies dropped after culture in heat-inactivated anti-Neu5Gc antibody-negative human serum, reducing binding of antibodies and complement from high-titer sera, while allowing maintenance of the undifferentiated state. Complete elimination of Neu5Gc would be likely to require using human serum with human feeder layers, ideally starting with fresh HESC that have never been exposed to animal products.     

In vitro allogeneic cytotoxicity in the solitary urochordate Styela clava.

Hemocytes from the solitary urochordate Styela clava can effect allogeneic cytotoxicity in vitro. Spectrophotometric and microscopic quantification of eosin-y dye exclusion revealed significantly greater frequencies of cell death in allogeneic hemocyte cultures when compared to autogeneic controls. This cytotoxic response was characterized by 1) transient activity such that specific cytotoxicity could be detected for only 4 hours of culture though continued specific killing may have been obscured by spontaneous cell death; 2) a necessity for cellular interaction demonstrated by the elimination of allogeneic cytotoxicity in the absence of cell contact; 3) killing of multiple targets by effector cells due to high levels of response at low allogeneic ratios; 4) insensitivity to altered temperature; 5) increased cytotoxicity in the absence of autologous plasma; 6) an absence of xenogeneic reactivity; 7) the presence of three hierarchical levels (low, intermediate, and high) of response. These data reflect events involved in the recognition of allogeneic cellular determinants resulting in specific cytotoxicity effected by immunocompetent cells. Such in vitro recognition and cytotoxic recognition and cytotoxic reactivity may be responsible for adaptive reactions caused by histoincompatibility in solitary tunicates.     

Sensitive and Specific Detection of the Non-Human Sialic Acid N-Glycolylneuraminic Acid In Human Tissues and Biotherapeutic Products

Background

Humans are genetically defective in synthesizing the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc), but can metabolically incorporate it from dietary sources (particularly red meat and milk) into glycoproteins and glycolipids of human tumors, fetuses and some normal tissues. Metabolic incorporation of Neu5Gc from animal-derived cells and medium components also results in variable contamination of molecules and cells intended for human therapies. These Neu5Gc-incorporation phenomena are practically significant, because normal humans can have high levels of circulating anti-Neu5Gc antibodies. Thus, there is need for the sensitive and specific detection of Neu5Gc in human tissues and biotherapeutic products. Unlike monoclonal antibodies that recognize Neu5Gc only in the context of underlying structures, chicken immunoglobulin Y (IgY) polyclonal antibodies can recognize Neu5Gc in broader contexts. However, prior preparations of such antibodies (including our own) suffered from some non-specificity, as well as some cross-reactivity with the human sialic acid N-acetylneuraminic acid (Neu5Ac).

Methodology/Principal Findings

We have developed a novel affinity method utilizing sequential columns of immobilized human and chimpanzee serum sialoglycoproteins, followed by specific elution from the latter column by free Neu5Gc. The resulting mono-specific antibody shows no staining in tissues or cells from mice with a human-like defect in Neu5Gc production. It allows sensitive and specific detection of Neu5Gc in all underlying glycan structural contexts studied, and is applicable to immunohistochemical, enzyme-linked immunosorbent assay (ELISA), Western blot and flow cytometry analyses. Non-immune chicken IgY is used as a reliable negative control. We show that these approaches allow sensitive detection of Neu5Gc in human tissue samples and in some biotherapeutic products, and finally show an example of how Neu5Gc might be eliminated from such products, by using a human cell line grown under defined conditions.

Conclusions

We report a reliable antibody-based method for highly sensitive and specific detection of the non-human sialic acid Neu5Gc in human tissues and biotherapeutic products that has not been previously described.     

Coexpression of CMP-sialic acid transporter reduces N-glycolylneuraminic acid levels of recombinant glycoproteins in Chinese hamster ovary cells

Recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells contain two forms of sialic acids; N-acetylneuraminic acid (Neu5Ac) as a major type and N-glycolylneuraminic acid (Neu5Gc) as a minor type. The Neu5Gc glycan moieties in therapeutic glycoproteins can elicit immune responses because they do not exist in human. In the present work, to reduce Neu5Gc levels of recombinant glycoproteins from CHO cell cultures, we coexpressed cytidine-5′-monophosphate-sialic acid transporter (CMP-SAT) that is an antiporter and transports cytosolic CMP-sialic acids (both forms) into Golgi lumen. When human erythropoietin was used as a target human glycoprotein, coexpression of CMP-SAT resulted in a significant decrease of Neu5Gc level by 41.4% and a notable increase of Neu5Ac level by 21.2%. This result could be reasonably explained by our hypothesis that the turnover rate of Neu5Ac to Neu5Gc catalyzed by CMP-Neu5Ac hydroxylase would be reduced through facilitated transportation of Neu5Ac into Golgi apparatus by coexpression of CMP-SAT. We confirmed the effects of CMP-SAT coexpression on the decrease of Neu5Gc level and the increase of Neu5Ac level using another glycoprotein human DNase I. Therefore, CMP-SAT coexpression might be an effective strategy to reduce the levels of undesired Neu5Gc in recombinant therapeutic glycoproteins from CHO cell cultures.     

Osmolality as a lever to modulate the N-glycolylneuraminicacid (Neu5Gc) level of a recombinant glycoprotein produced in Chinese hamster ovary cells

Glycoproteins could be highly sialylated, and controlling the sialic acid levels for some therapeutic proteins is critical to ensure product consistency and efficacy. N-acetylneuraminic acid (Neu5Ac, or NANA) and N-glycolylneuraminic acid (Neu5Gc, or NGNA) are the two most common forms of sialic acids produced in mammalian cells. As Neu5Gc is not produced in humans and can elicit immune responses, minimizing Neu5Gc formation is important in controlling this quality attribute for complex glycoproteins. In this study, a sialylated glycoprotein was used as the model molecule to study the effect of culture osmolality on Neu5Gc. A 14-day fed-batch process with osmolality maintained at physiological levels produced high levels of Neu5Gc. Increase of culture osmolality reduced the Neu5Gc level up to 70–80%, and the effect was proportional to the osmolality level. Through evaluating different osmolality conditions (300–450 mOsm/kg) under low or high pCO₂, we demonstrated that osmolality could be an effective process lever to modulate the Neu5Gc level. Potential mechanism of osmolality impact on Neu5Gc is discussed and is hypothesized to be cytosol NADH availability related. Compared with cell line engineering efforts, this simple process lever provides the opportunity to readily modulate the Neu5Gc level in a cell culture environment.     

Biosynthesis of N-glycolyneuraminic acid. The primary site of hydroxylation of N-acetylneuraminic acid is the cytosolic sugar nucleotide pool

N-Glycolylneuraminic acid (Neu5Gc) is an oncofetal antigen in humans and is developmentally regulated in rodents. We have explored the biology of N-acetylneuraminic acid hydroxylase, the enzyme responsible for conversion of the parent sialic acid, N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. We show that the major sialic acid in all compartments of murine myeloma cell lines is Neu5Gc. Pulse-chase analysis in these cells with the sialic acid precursor [6-3H]N-acetylmannosamine demonstrates that most of the newly synthesized Neu5Gc appears initially in the cytosolic low-molecular weight pool bound to CMP. The percentage of Neu5Gc on membrane-bound sialic acids closely parallels that in the CMP-bound pool at various times of chase, whereas that in the free sialic acid pool is very low initially, and rises only later during the chase. This implies that conversion from Neu5Ac to Neu5Gc occurs primarily while Neu5Ac is in its sugar nucleotide form. In support of this, the hydroxylase enzyme from a variety of tissues and cells converted CMP-Neu5Ac to CMP-Neu5Gc, but showed no activity towards free or alpha-glycosidically bound Neu5Ac. Furthermore, the majority of the enzyme activity is found in the cytosol. Studies with isolated intact Golgi vesicles indicate that CMP-Neu5Gc can be transported and utilized for transfer of Neu5Gc to glycoconjugates. The general properties of the enzyme have also been investigated. The Km for CMP-Neu5Ac is in the range of 0.6-2.5 microM. No activity can be detected against the beta-methylglycoside of Neu5Ac. On the other hand, inhibition studies suggest that the enzyme recognizes both the 5'-phosphate group and the pyrimidine base of the substrate. Taken together, the data allow us to propose pathways for the biosynthesis and reutilization of Neu5Gc, with initial conversion from Neu5Ac occurring primarily at the level of the sugar nucleotide. Subsequent release and reutilization of Neu5Gc could then account for the higher steady-state level of Neu5Gc found in all of the sialic acid pools of the cell.     

Comparison of theN-glycoloylneuraminic andN-acetylneuraminic acid content of platelets and their precursors using high performance anion exchange chromatography

N-Acetylneuraminic acid (Neu5Ac) andN-glycoloylneuraminic acid (Neu5Gc) are distributed widely in nature. Using a Carbopac PA-1 anion exchange column, we have determined the ratios of Neu5Ac and Neu5Gc in hydrolysates of platelets and their precursors: a rat promegakaryoblastic (RPM) cell line and a human megakaryoblastic leukemia cell line (MEG-01). The ratio of Neu5Gc:Neu5Ac in cultured RPM cells is 16:1, whereas in platelet rich plasma and cultured MEG-01 cells it is 1:38 and 1:28, respectively. The nature of these sialic acids from RPM cells was verified using thin layer chromatography and liquid secondary ion mass spectrometry. The relevance of increased Neu5Gc levels in early stages of development is discussed.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - RPM rat promegakaryoblast - MEG-01 human megakaryoblastic leukaemia cell line - PAD pulsed amperometric detection - WGA wheat germ agglutinin - FCS foetal calf serum - PPEADF phosphatidylethanolamine dipalmitoyl - LSIMS liquid secondary ion mass spectrometry - HPAEC high performance anion exchange chromatography - TBA thiobarbituric acid     

Effects of culture conditions on N‐glycolylneuraminic acid (Neu5Gc) content of a recombinant fusion protein produced in CHO cells

CHO cells express glycoproteins containing both the N‐acetylneuraminic acid (Neu5Ac) and minor amounts of the N‐glycolylneuraminic acid (Neu5Gc) forms of sialic acid. As Neu5Gc is not expressed in humans and can be recognized as a foreign epitope, there is the potential for immunogenicity issues for glycoprotein therapeutics. During process development of a glycosylated fusion protein expressed by CHO cells, a number of culture conditions were identified that affected the Neu5Gc content of the recombinant glycoprotein. Sodium butyrate (SB), a well‐known additive reported to enhance recombinant protein productivity in specific cases, minimally affected product titers here, but did decrease Neu5Gc levels by 50–62%. A shift in culture temperature to a lower value after the exponential growth phase was used to extend the culture period. It was found that the Neu5Gc levels were 59% lower when the temperature shift occurred later near the stationary phase of the culture compared to an early‐temperature shift, near the end of the exponential growth phase. Studies on the effects of pCO₂ with this product showed that the Neu5Gc levels were 46% lower at high pCO₂ conditions (140 mmHg) compared to moderate pCO₂ levels (20–80 mmHg). Finally, a comparison of sodium carbonate versus sodium hydroxide as the base used for pH control resulted in a reproducible 33% decrease in Neu5Gc in bioreactors using sodium hydroxide. These results are of practical importance as SB is a commonly tested additive, and the other factors affecting Neu5Gc can conveniently be used to reduce or control Neu5Gc in processes for the manufacture of glycoprotein therapeutics. Biotechnol. Bioeng. 2010;105: 1048–1057. © 2009 Wiley Periodicals, Inc.     

A structural difference between the cell surfaces of humans and the great apes

The sialic acids are major components of the cell surfaces of animals of the deuterostome lineage. Earlier studies suggested that humans may not express N-glycolyl-neuraminic acid (Neu5Gc), a hydroxylated form of the common sialic acid N-acetyl-neuraminic acid (Neu5Ac). We find that while Neu5Gc is essentially undetectable on human plasma proteins and erythrocytes, it is a major component in all the four extant great apes (chimpanzee, bonobo, gorilla and orangutan) as well as in many other mammals. This marked difference is also seen amongst cultured lymphoblastoid cells from humans and great apes, as well as in a variety of other tissues compared between humans and chimpanzees, including the cerebral cortex and the cerebrospinal fluid. Biosynthetically, Neu5Gc arises from the action of a hydroxylase that converts the nucleotide donor CMP-Neu5Ac to CMP-Neu5Gc. This enzymatic activity is present in chimpanzee cells, but not in human cells. However, traces of Neu5Gc occur in some human tissues, and others have reported expression of Neu5Gc in human cancers and fetal tissues. Thus, the enzymatic capacity to express Neu5Gc appears to have been suppressed sometime after the great ape-hominid divergence. As terminal structures on cell surfaces, sialic acids are involved in intercellular cross-talk involving specific vertebrate lectins, as well as in microbe-host recognition involving a wide variety of pathogens. The level of sialic acid hydroxylation (level of Neu5Ac versus Neu5Gc) is known to positively or negatively affect several of these endogenous and exogenous interactions. Thus, there are potential functional consequences of this widespread structural change in humans affecting the surfaces of cells throughout the body. Am J Phys Anthropol 107:187-198, 1998. © 1998 Wiley-Liss, Inc.     

Chemoenzymatic synthesis and characterization of N-glycolylneuraminic acid-carrying sialoglycopolypeptides as effective inhibitors against equine influenza virus hemagglutination

A series of novel sialoglycopolypeptides carrying N-glycolylneuraminic acid (Neu5Gc)-containing trisaccharides having α(2 → 3)- and α(2 → 6)-linkages in the side chains of γ-polyglutamic acid (γ-PGA) were designed as competitive inhibitors against equine influenza viruses (EIV), which critically recognize the Neu5Gc residue for receptor binding. Using horse red blood cells (HRBC) we successfully evaluated the binding activity of the multivalent Neu5Gc ligands to both equine and canine influenza viruses in the hemagglutination inhibition (HI) assay. Our findings show the multivalent α2,3-linked Neu5Gc-ligands (3a–c and 7) selectively inhibit hemagglutination mediated by both influenza viruses and display a strong inhibitory activity. Our results indicate that the multivalent Neu5Gc-ligands can be used as novel probes to elucidate the mechanism of infection/adhesion of Neu5Gc-binding influenza viruses.     

Determination of sialic acids in immune system cells (coelomocytes) of sea urchin,Paracentrotus lividus,using capillary LC-ESI-MS/MS

Coelomocytes are considered to be immune effectors of sea urchins. Coelomocytes are the freely circulating cells in the body fluid contained in echinoderm coelom and mediate the cellular defence responses to immune challenges by phagocytosis, encapsulation, cytotoxicity and the production of antimicrobial agents. Coelomocytes have the ability to recognize self from non-self. Considering that sialic acids play important roles in immunity, we determined the presence of sialic acid types in coelomocytes of Paracentrotus lividus. Homogenized coelomocytes were kept in 2 M aqueous acetic acid at 80 °C for 3 h to liberate sialic acids. Sialic acids were determined by derivatization with 1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride (DMB) followed by capillary liquid-chromatography-electrospray ionization/tandem mass spectrometry (CapLC-ESI-MS/MS). Standard sialic acids; Neu5Ac, Neu5Gc, KDN and bovine submaxillary mucin showing a variety of sialic acids were used to confirm sialic acids types. We found ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac₂, Neu5,7Ac₂, Neu5,8Ac₂, Neu5,7,9Ac₃, Neu5Gc7,9Ac₂, Neu5Gc7Ac) isolated in limited amounts from total coelomocyte population. Neu5Gc type of sialic acids in coelomocytes was the most abundant type sialic acid when compared with other types. This is the first report on the presence of sialic acid types in coelomocytes of P. lividus using CapLC-ESI-MS/MS-Ion Trap system (Capillary Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry).     

Chemo-enzymatic synthesis of the carbohydrate antigen N-glycolylneuraminic acid from glucose

N-Glycolylneuraminic acid (Neu5Gc) is a non-human sialic acid, which may play a significant role in human pathologies, such as cancer and vascular disease. Further studies into the role of Neu5Gc in human disease are hindered by limited sources of this carbohydrate. Using a chemo-enzymatic approach, Neu5Gc was accessed in six steps from glucose. The synthesis allows access to gram-scale quantities quickly and economically and produces Neu5Gc in superior quality to commercial sources. Finally, we demonstrate that the synthesized Neu5Gc can be incorporated into the cell glycocalyx of human cells, which do not naturally synthesize this sugar. The synthesis produces Neu5Gc suitable for in vitro or in vivo use.     

Targeting FcαRI on polymorphonuclear cells induces tumor cell killing through autophagy

Neutrophils are the most abundant circulating FcR-expressing WBCs with potent cytotoxic ability. Currently, they are recognized as promising effector cells for Ab-mediated immunotherapy of cancer, because their capacity to kill tumor cells is greatly enhanced by tumor Ag-specific mAbs. The FcαRI represents the most potent FcR on neutrophils for induction of Ab-mediated tumor cell killing. However, the mechanisms of cell death that are induced are poorly understood. Because these mechanisms can be used for modulation of anticancer treatment, we investigated the tumor cell death induced by neutrophil-mediated Ab-dependent killing via FcαRI. Human mammary carcinoma cells were efficiently killed when incubated with human neutrophils and tumor-specific FcαRI bispecific or IgA Abs. Interestingly, we observed characteristics of autophagy such as autophagic structures by electron microscopy and LC3B(+) autophagosomes in different human epithelial carcinoma cells, which resulted in tumor cell death. To a lesser extent, necrotic features, such as cellular membrane breakdown and spillage of intracellular content, were found. By contrast, apoptotic features including fragmented nuclei, Annexin V-positivity, and presence of cleaved caspase-3 were not observed. These findings indicate that neutrophils mainly facilitate autophagy to induce tumor cell death rather than the more commonly recognized apoptotic cell death mechanisms induced by NK cells or cytotoxic T cells. This knowledge not only reveals the type of tumor cell death induced in neutrophil-mediated, Ab-dependent cellular cytotoxicity, but importantly opens up additional perspectives for modulation of anticancer therapy in, for example, apoptosis-resistant tumor cells.     

Mechanism of uptake and incorporation of the non-human sialic acid N-glycolylneuraminic acid into human cells

N-Glycolylneuraminic acid (Neu5Gc) is a widely expressed sialic acid in mammalian cells. Although humans are genetically deficient in producing Neu5Gc, small amounts are present in human cells in vivo. A dietary origin was suggested by human volunteer studies and by observing that free Neu5Gc is metabolically incorporated into cultured human carcinoma cells by unknown mechanisms. We now show that free Neu5Gc uptake also occurs in other human and mammalian cells. Inhibitors of certain non-clathrin-mediated endocytic pathways reduce Neu5Gc accumulation. Studies with human mutant cells show that the lysosomal sialic acid transporter is required for metabolic incorporation of free Neu5Gc. Incorporation of glycosidically bound Neu5Gc from exogenous glycoconjugates (relevant to human gut epithelial exposure to dietary Neu5Gc) requires the transporter as well as the lysosomal sialidase, which presumably acts to release free Neu5Gc. Thus, exogenous Neu5Gc reaches lysosomes via pinocytic/endocytic pathways and is exported in free form into the cytosol, becoming available for activation and transfer to glycoconjugates. In contrast, N-glycolylmannosamine (ManNGc) apparently traverses the plasma membrane by passive diffusion and becomes available for conversion to Neu5Gc in the cytosol. This mechanism can also explain the metabolic incorporation of chemically synthesized unnatural sialic acids, as reported by others. Finally, to our knowledge, this is the first example of delivery to the cytosol of an extracellular small molecule that cannot cross the plasma membrane, utilizing fluid pinocytosis and a specific lysosomal transporter. The approach could, thus, potentially be generalized to any small molecule that has a specific lysosomal transporter but not a plasma membrane transporter.     

Distribution and localization of CMP-N-acetylneuraminic acid hydroxylase and N-glycolylneuraminic acid-containing glycoconjugates in porcine lymph node and peripheral blood lymphocytes

An immunohistochemical analysis was performed on paraplast-embedded sections of porcine lymph node with antibodies specific for CMP-N-acetylneuraminic acid hydroxylase (h-3 antibody) and glycoconjugate-bound N-glycolylneuraminic acid (Neu5Gc), which appears as a result of the hydroxylase reaction (a-Gc antibody). The observed localization of the enzyme in cells of the perifollicular zone, including lymphocytes, was reflected in a similar distribution of glycoconjugate-bound Neu5Gc. This result confirms previous biochemical investigations on the role of the hydroxylase in regulating Neu5Gc biosynthesis in vitro on a histological level. An analysis of lymphocytes isolated from porcine thymus, spleen, lymph node and peripheral blood revealed differences in the amount of Neu5Gc in the various lymphocytes that correlated well with the activity of the hydroxylase determined in these cells. The largest amount of Neu5Gc and highest activity of the enzyme were detected in the peripheral blood lymphocytes (PBL). Immunohistochemical studies with a-Gc and h-3 antibodies on sections of paraplast-embedded PBL showed that these antigens were located at the cell surface and in the cytosol, respectively. Ultrastructural immunocytochemistry with the h-3 antibody and immunogold labelling was used to investigate the subcellular localization of the hydroxylase. The enzyme was detected in the cytosol in the vicinity of the nuclear membrane and the outer membrane of mitochondria, in particular those close to the nucleus. The antigen was also detected on cytoplasmic tubular structures. In addition, a weak labelling of the Golgi apparatus was also observed occasionally. The possibility that this localization may be related to the availability of the substrate CMP-Neu5Ac and the redox partner cytochrome b5 is discussed.     

Paul-Bunnell antigen and a possible mechanism of formation of heterophile antibodies in patients with infectious mononucleosis

Sera of patients with infectious mononucleosis contain heterophile anti-Paul- Bunnell (PB) antibodies to erythrocytes of numerous mammalian species. Evidence is presented that the corresponding antigen of bovine erythrocytes is not, as previously described, a single molecule, but a series of glycoproteins with glycans terminated with N-glycolylneuraminic acid (Neu5Gc). The latter compound should be an important part of the PB epitope because, in agreement with the results of others, we found that desialylation of the PB antigen abolishes almost completely its activity. We examined three different preparations of GM3 ganglioside for their capacity to bind anti-PB and found that only GM3 from horse erythrocytes containing Neu5Gc exhibited a low although ELISA measurable PB activity. The other two GM3 preparations, from bovine milk and dog erythrocytes, containing N-acetylneuraminic acid (Neu5Ac) bound little if any anti-PB antibodies. This finding confirms a previous report that human erythrocyte Neu5Ac containing sialoglycoprotein with similar O-linked glycans as the PB-antigen of bovine erythrocytes exhibits only very low PB activity (Patarca & Fletcher, 1995, Crit Rev Oncogen., 6: 305). In conclusion, we present a hypothesis that anti-PB antibodies in patients with infectious mononucleosis are formed against infection-induced cell membrane glycoconjugates containing highly immunogenic Neu5Gc.     

The role of CMP-N-acetylneuraminic acid hydroxylase in determining the level of N-glycolylneuraminic acid in porcine tissues

The biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc) occurs by the action of cytidine monophosphate-N-acetylneuraminate (CMP-Neu5Ac) hydroxylase. Previous investigations on a limited number of tissues suggest that the activity of this enzyme governs the extent of glycoconjugate sialylation with Neu5Gc. Using improved analytical procedures and a panel of nine porcine tissues, each expressing different amounts of Neu5Gc, we have readdressed the issue of the regulation of Neu5Gc incorporation into glycoconjugates. The following parameters were measured for each tissue: the molar ratio Neu5Gc/Neu5Ac, the activity of the hydroxylase, and the relative amount of hydroxylase protein, as determined by enzyme-linked immunosorbent assay (ELISA). A positive correlation between the activity of the hydroxylase and the molar ratio Neu5Gc/Neu5Ac was observed for each tissue. In addition, the hydroxylase activity correlated with the amount of enzyme protein, though in heart and lung disproportionately large amounts of immunoreactive protein were detected. Taken together, the results suggest that the incorporation of Neu5Gc into glycoconjugates is generally controlled by the amount of hydroxylase protein expressed in a tissue.