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

Abstract

Biosynthesis of the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) was lost during human evolution due to inactivation of the CMAH gene, possibly expediting divergence of the Homo lineage, due to a partial fertility 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 glycoconjugate products. Partially N-glycolylated-CS was chemically synthesized as a standard for mass spectrometry to confirm 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.

Keywords: evolution; glycobiology; sialic acid.

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 1.14.18.2) (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 4.1.3.3) (4, 50, 51). (ii) Epimerization of ManNGc to GlcNGc can be achieved by GlcNAc-2′-epimerase (EC 5.1.3.8) (4). (iii) Phosphorylation of GlcNGc in the 6′ position to result in GlcNGc-6P’s beings catalyzed by GlcNAc kinase (EC 2.7.1.59) (4). (iv) Conversion of GlcNGc 6-P to GlcNGc 1-P might be catalyzed by GlcNAc 6-P phosphomutase (EC 5.4.2.3) (52). (v) GlcNGc 1-P would thereafter react with UTP to form UDP-GlcNAc, a reaction potentially catalyzed by UDP-N-acetylglucosamine diphosphorylase (EC 2.7.7.23) (53). (vi) Epimerization of UDP-GlcNGc to UDP-GalNGc is catalyzed by UDP-GlcNAc 4-epimerase (EC 5.1.3.7) (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.

Fig. 2.

Natural occurrence of UDP-N-glycolyhexosamines in mouse liver. Nucleoside diphospho sugars were isolated from deproteinized WT and Cmah^−/− mouse liver homogenates via C-18 and charcoal columns. Samples were further treated with phosphatase following DEAE sephacel cleanup before LC-MS analysis. (A) Chromatogram of WT mouse liver nucleoside diphospho sugars with an underlying mass of UDP-HexNAc (606 Da) and (B) MS analysis of the respective peak fraction. (C) Chromatogram of Cmah^−/− mouse liver nucleoside diphospho sugars with an underlying mass of UDP-HexNAc (606 Da) and (D) MS analysis of the respective peak fraction. (E) Chromatogram of WT mouse liver nucleoside diphospho sugars with an underlying mass of UDP-HexNGc (622 Da) and (F) MS analysis of the respective peak fraction. (G) chromatogram of Cmah^−/− mouse liver nucleoside diphospho sugars with an underlying mass of UDP-HexNGc (622 Da) and (H) MS analysis of the theoretical peak fraction for the elution time at which UDP-HexNGc would elute if present.

Fig. S1.

Synthesis of partially N-glycolylated CS. (A) Schematic representation of the procedure to generate partially N-glycolylated CS from commercial-grade shark cartilage CS. (B) The extracted ion current chromatograph for [¹³C₆]aniline-labeled nonmodified singly sulfated disaccharide residues present after chemical modification. The relative abundances of both D0a4 and D0a6 are consistent with unmodified starting material. (C) The extracted ion current chromatograph for the corresponding [¹³C₆]aniline-labeled glycolylated disaccharides D0q4 and D0q6. (D) The product ion mass spectrum for D0q4. (E) The product ion mass spectrum for D0q6.

Fig. 3.

Detection of N-glycolyl groups in CS isolated from WT and Cmah^−/− liver and lamb muscle. (A) SRM for ^0,2X₁ product ion from D0q4 (m/z = 435) present in WT mouse liver. The y axis represents ion intensity, 0–1,000 arbitrary units. (B) CID mass spectrum of D0q4 detected in WT mouse liver. (C) SRM for ^0,2X₁ product ion from D0q4 present in Cmah^−/− mouse liver. (D) SRM for ^0,2X₁ product ion from D0q4 (m/z = 435) present in normal lamb muscle. The y axis represents ion intensity, 0–100 arbitrary units. (E) CID mass spectrum of D0q4 present in lamb muscle.

Fig. 4.

LC/MS analysis of serum from primates and mice. (A and B) The extracted ion current (XIC) for D0q4 in chimpanzee serum identified by retention time and tandem MS (CID spectrum) compared with D0q4 standard. A 35-min gradient was used for this sample. (C and D) The XIC and corresponding CID spectrum for small trace amounts of D0q4 found in human serum. A 35-min gradient was used for this sample. (E and F) The XIC and corresponding CID spectrum for D0q4 found in a WT mouse. A 100-min gradient with higher resolution was used for this sample. (G and H) The XIC and corresponding CID spectrum for the time range where D0q4 normally elutes (arrow) in the 100-min gradient used for this sample. (I and J) The XIC and CID spectrum for D0q4 detected in Cmah^−/− mice fed a Neu5Gc-containing diet. A 35-min gradient was used for this sample. The asterisks and double asterisks in the mouse XIC traces are isobaric species that are detected along with D0q4 but do not coelute with D0q4 standard. The asterisks in the CID spectra denote a daughter ion that does not correspond to a product ion from D0q4 standard.

Fig. 5.

Detection of N-glycolyl groups in CS isolated from partially mineralized fossil samples. (A) The accumulative extracted ion current chromatograph for Y₁, ^0,2X₁, and M-HSO₃ daughter ions consistent with N-glycolylated disaccharide residues (m/z = 393, 435 and 471, respectively) for fossil F44. (B) The product ion mass spectrum of D0q4 (m/z value = 551). (C) CS compositional analysis comparing D0a4 and D0a6 abundance of bone and fossil samples expressed as molar percent (mol %).

Fig. S2.

Disaccharide compositional analyses of CS. Relative abundance of CS disaccharides in contemporary (human and chimpanzee bone) compared with fossil specimens expressed as molar percent (mol %).

Fig. S3.

Disaccharide compositional analyses of HS. Relative abundance of HS disaccharides in contemporary (human and chimpanzee bone) compared with fossil specimens expressed as molar percent (mol %).

Fig. 6.

Potential scenario for the role of Neu5Gc loss and female anti-Neu5Gc immunity in the origin of the genus Homo via interplay of natural and sexual selection acting on cell-surface Sias. There are many known pathogens that recognize and exploit Neu5Gc (blue diamond) as a receptor on host target cells (9). Natural selection by such pathogens may have selected for rare CMAH null alleles that abolish Neu5Gc expression in homozygote individuals (12). Such individuals have only Neu5Ac and its derivatives on their cells (red diamonds) allowing an escape from pathogens, but at higher frequencies would be targeted by adapting pathogens, resulting in maintenance of a balanced polymorphism. CMAH^−/− females with anti-Neu5Gc antibodies also present in their reproductive tract would favor sperm from CMAH^−/− males due to anti-Neu5Gc antibody-mediated cryptic selection against CMAH^+/− or CMAH^+/+ males expressing Neu5Gc on their sperm. Once the frequency of the CMAH null allele reaches a critical level, this process can drive fixation of the loss-of-function allele in a population by directional selection (12).