Sialiinimutatio ihmistymisen selittäjänä

Risto Koivula, 11/6/2020 11:13:59 PM, 403855

N-glycolyl groups of nonhuman chondroitin sulfates survive in ancient fossils

Anne K. Bergfeld, Roger Lawrence, Sandra L. Diaz, Oliver M. T. Pearce, Darius Ghaderi, Pascal Gagneux, Meave G. Leakey, and Ajit Varki

PNAS first published September 11, 2017;

Edited by Chi-Huey Wong, Academia Sinica, Taipei, Taiwan, and approved July 25, 2017 (received for review April 19, 2017)


We identified a glycan modification called N-glycolylated chondroitin sulfate (Gc-CS), derived from metabolic turn- over of the nonhuman sialic acid N-glycolylneuraminic acid (Neu5Gc).The presence of Gc-CS could be demonstra- ted in species rich in Neu5Gc using chemically synthesized Gc-CS as a standard for mass spectrometry. Although humans cannot synthesize Neu5Gc due to a loss-of-function mutation, trace amounts of Gc-CS were found in humans, apparently derived from Neu5Gc-containing foods. Gc-CS was more easily detectable in animal fossils as old as 4 My, allowing indirect fossil evidence of Neu5Gc expression. These findings enable future studies to date the loss of Neu5Gc biosynthesis during human evolution and investigate this glycosaminoglycan modification in humans who consume Neu5Gc-rich foods (red meats).


Biosynthesis of the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) was lost during human evo- lution due to inactivation of the CMAH gene,possibly expediting divergence of the Homo lineage, due to a partial fer- tility barrier.Neu5Gc catabolism generates N-glycolylhexosamines,which are potential precursors for glycoconjugate biosynthesis. We carried out metabolic labeling experiments and studies of mice with human-like Neu5Gc deficiency to show that Neu5Gc degradation is the metabolic source of UDP-GlcNGc and UDP-GalNGc and the latter allows an unexpectedly selective incorporation of N-glycolyl groups into chondroitin sulfate (CS) over other potential glycocon- jugate products.Partially N-glycolylated-CS was chemically synthesized as a standard for mass spectrometry to con- firm its natural occurrence. Much lower amounts of GalNGc in human CS can apparently be derived from Neu5Gc-containing foods, a finding confirmed by feeding Neu5Gc-rich chow to human-like Neu5Gc-deficient mice. Unlike the case with Neu5Gc, N-glycolyl-CS was also stable enough to be detectable in animal fossils as old as 4 My. This work opens the door for investigating the biological and immunological significance of this glycosaminoglycan modification and for an “ancient glycans” approach to dating of Neu5Gc loss during the evolution of Homo.

All vertebrate cells are covered with a complex array of glycoconjugates, with sialic acids (Sias) typically occupying terminal positions of many glycan chains (1). The two most common Sias in most mammals are N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), which differ by a single oxygen atom (1). The only known metabolic pathway for Neu5Gc biosynthesis is hydroxylation of its precursor Neu5Ac, catalyzed by the enzyme CMP-Neu5Ac hydroxylase (CMAH) (2⇓–4). A loss-of-function mutation in the corresponding single-copy CMAH gene is fixed in humans, who can no longer biosynthesize Neu5Gc (3, 5, 6). Multiple methods of genomic analysis estimated that the loss of CMAH function occurred ∼2–3 Mya in the hominin lineage (7, 8).

There are many known and possible biological consequences of human Neu5Gc loss, discussed in detail elsewhere (9). Following loss of Neu5Gc biosynthesis, the human immune system also recognizes Neu5Gc-bearing glycans as foreign and antigenic molecules (10, 11). In human-like Cmah−/− Neu5Gc-deficient mice, the resulting anti-Neu5Gc antibodies could mediate female intrauterine immune selection against sperm from Cmah-positive WT males (12, 13). Models of frequency-dependent selection regimes showed that such a reproductive incompatibility by female immunity could drive this loss-of-function allele to fixation after it reached moderate initial frequencies. We therefore postulated that fixation of the CMAH null state in the hominin lineage leading to our species could have expedited divergence of the genus Homo ∼2–3 Mya (12). However, there is as yet no direct proof for this hypothesis.

Notably, human cellular pathways still allow metabolic incorporation of scavenged Neu5Gc into endogenous cellular glycans (4, 10, 14). This process can even occur in intact humans, by metabolically incorporating trace amounts of Neu5Gc from mammal-derived food products, particularly red meats (beef, lamb, and pork). Incorporation of trace amounts of such exogenous Neu5Gc into human cell-surface structures in the face of an anti-Neu5Gc antibody response makes Neu5Gc the first known “xeno-autoantigen” in humans (15⇓–17). However, incorporation levels are far lower than endogenous levels in WT (Cmah intact) mice or in chimpanzees, our closest living evolutionary relatives (18).

As an alternative to activation to CMP-Neu5Gc and incorporation into glycoconjugates, Neu5Gc can also follow a degradative metabolic route, resulting in loss of the N-glycolyl group and formation of glucosamine 6-phosphate (4). One intermediate on this multistep pathway was found to be N-glycolylglucosamine (GlcNGc) (4) (Fig. 1A).

Additionally, mammalian cells cultured in synthetic GlcNGc were found to biosynthesize UDP-GlcNGc, which was incorporated as O-GlcNGc modifications of proteins (19) and was also detected in O-glycans, likely as N-glycolyl- galactosamine (GalNGc) (20, 21). Existence of HexNGcs in O-glycans and N-glycans was also suggested after analyzing cells supplemented with synthetic ManNGc (22). In addition, we found that culturing mammalian cells in synthetic GalNGc gave rise to UDP-GalNGc and UDP-GlcNGc, which serve as sugar nucleotide donors for incor- poration into cellular O-glycans, gangliosides, heparan sulfates (HS), and chondroitin sulfates (CS) (23). The latter two glycan classes are major components of extracellular matrix and bone (24). While cellular pathways allowed low-level incorporation of artificially provided GalNGc or GlcNGc into most major glycan classes, we noted that the N-glycolyl group was most prominently found in CS (23). Here we explore the possibility that metabolic turnover of naturally occurring and/or food-derived Neu5Gc might result in a so-far-unnoticed subset of cellular glycans that naturally contain HexNGc residues. We also apply these findings to challenging questions regarding indirect fossil evidence of Neu5Gc expression over a time span of millions of years.

Fig. 1.

Possible underlying mechanism for the natural occurrence of HexNGc in animal glycoconjugates.

(A) The single known source for N-glycolyl groups in animals is the conversion of the N-acetyl group of CMP-Neu5Ac to an N-glycolyl group in CMP-Neu5Gc, which is catalyzed by Cmah (EC (49). Therewith, N-glycolhexosamines are to be Neu5Gc derivatives. Based on the well-studied N-acetylhexosamine pathways in animals we suggest a metabolic route to result in glycoconjugates comprising GlcNGc and GalNGc in nature.

(i) Conversion of Neu5Gc into ManNGc is catalyzed by the N-acetylneuraminate lyase (EC (4, 50, 51).

(ii) Epimerization of ManNGc to GlcNGc can be achieved by GlcNAc-2′-epimerase (EC (4).

(iii) Phosphorylation of GlcNGc in the 6′ position to result in GlcNGc-6P’s beings catalyzed by GlcNAc kinase (EC (4).

(iv) Conversion of GlcNGc 6-P to GlcNGc 1-P might be catalyzed by GlcNAc 6-P phosphomutase (EC (52).

(v) GlcNGc 1-P would thereafter react with UTP to form UDP-GlcNAc, a reaction potentially catalyzed by UDP-N-acetylglucosamine diphosphorylase (EC (53).

(vi) Epimerization of UDP-GlcNGc to UDP-GalNGc is catalyzed by UDP-GlcNAc 4-epimerase (EC (54). UDP-GlcNGc and UDP-GalNGc then serve as precursors for glycan assembly.

(B) Human THP-I cells and

(C) CHO LEC29.lec32 cells were cultured in the presence of [3H-glycolyl]Neu5Gc. Desalted GAGs were divided into three samples, from which one sample remained untreated (filled gray), one sample was treated with chondroitinase ABC (black line), and the last sample was treated with heparinases (gray line). The disaccharides were separated from the intact GAG chains by gel filtration chromatography.

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Muokannut: Risto Koivula, 11/6/2020 11:45:53 PM

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