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2. antibodies against Neu5Gc, with the highest levels comparable to those of the previously known anti–galactose xenoreactive antibodies. This finding represents Tacalcitol monohydrate an instance wherein humans absorb and metabolically incorporate a nonhuman dietary component enriched in foods of mammalian origin, even while generating xenoreactive, and potentially autoreactive, antibodies against the same molecule. Potential implications for human diseases are briefly discussed. Sialic acids (Sias) are nine-carbon sugars typically found as outermost units on the mammalian cellular glycocalyx and on secreted glycoproteins (14). The most common Sias areN-acetylneuraminic acid (Neu5Ac) andN-glycolylneuraminic acid (Neu5Gc). Cellular Neu5Gc is generated by hydroxylation of the sugar nucleotide donor CMP-Neu5Ac to CMP-Neu5Gc, catalyzed by CMP-Neu5Ac hydroxylase (CMAH) (57). Although Neu5Gc is a major Sia in most mammals (including our closest evolutionary relatives, the great apes) (8), it is thought to be absent in healthy humans (13). Indeed, humans generate immune responses against i.v.-administered molecules carrying Neu5Gc, e.g., the serum sickness reaction to equine antithymocyte globulin therapy (9,10). These findings are explained by a human-specific inactivating mutation in theCMAHgene (1113) that occurred 2.5 million to 3 million years ago (14). Despite no known alternate pathway for Neu5Gc synthesis in humans, antibodies have been used to claim its presence in some human cancers (1520) and human fetal meconium (15). However, the specificity of the polyclonal antibodies used was not well defined. One study using mAbs failed to detect Neu5Gc in human tumors and tissues (21). However, these mAbs were specific for Neu5Gc only in the context of underlying structural motifs. Another mAb thought to be specific for Neu5Gc cross-reacts with some sulfated glycolipids (22). Meanwhile, some reports claim chemical proof for Neu5Gc in human tumors Rabbit Polyclonal to LSHR (18,19,23). Human biosynthetic pathways could theoretically allow exogenous Neu5Gc to be metabolically incorporated (13,24). Indeed, human cells cultured in FCS express cell surface Neu5Gc in small amounts (8,21). However, it is not known whether this expression represents passive adsorption of serum glycoconjugates or metabolic incorporation. Although earlier studies claimed the absence of Neu5Gc from normal human tissues, we noted a small HPLC peak at the elution time of Neu5Gc in extracts from human organs (8). Here we explore whether Neu5Gc is actually present in normal and/or pathological human tissues, if Tacalcitol monohydrate normal humans can take up Neu5Gc from dietary sources, and if we spontaneously express antibodies against it. == Materials and Methods == Western Blot Analysis.Serum proteins or total proteins extracted from Neu5Gc-fed or nonfed Caco-2 cells were either treated or sham-treated with 10 milliunits ofArthrobacter ureafacienssialidase (EY Laboratories) in 100 mM sodium acetate, pH 5.5, at 37C for 3 h. After SDS/PAGE, the separated proteins were transferred to nitrocellulose membrane, and binding of the anti-Neu5Gc antibody [1:10,000 in Tris-buffered saline with 0.1% Tween 20 (TBST)] was detected by using a Tacalcitol monohydrate secondary horseradish peroxidase-conjugated donkey anti-chicken IgY antibody (1:30,000 in TBST) and Supersignal West Pico (Pierce). Immunohistochemistry Using the Chicken Anti-Neu5Gc Antibody.Human tissues were collected by an approved Institutional Review Board protocol, from autopsies or unused pathological material, frozen in OCT compound (Sakura Finetek, Torrance, CA), and archived at 70C. Harold McClure (Yerkes Primate Center, Atlanta) generously provided frozen chimpanzee tissues. Frozen tissue sections were air-dried for 30 min and fixed in 10% buffered formalin for 30 min. Endogenous peroxidase activity was quenched, and nonspecific binding sites were blocked with 5% (Neu5Gc-free) human serum in PBS for 30 min. Sections were then incubated with the anti-Neu5Gc antibody in 5% human serum/PBS at a 1:200 dilution at room temperature for 2 h (seeSupporting Text, which is published as supporting information on the PNAS web site,www.pnas.org, for antibody production). After washing, horseradish peroxidase-conjugated donkey anti-chicken IgY antibody in 5% human serum/PBS at a 1:100 dilution was applied for 1 h. Control sections were incubated with normal IgY. Specific binding was detected by using the Nova Red substrate kit (Vector Laboratories), followed by hematoxylin counterstaining. Detection of Neu5Gc by HPLC Analysis and MS.Human autopsy tissues (0.5 g) were subjected.