N-glycolylneuraminic acid deficiency in mice: implications for human biology and evolution

Abstract

Humans and chimpanzees share >99% identity in most proteins. One rare difference is a human-specific inactivating deletion in the CMAH gene, which determines biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc). Since Neu5Gc is prominent on most chimpanzee cell surfaces, this mutation could have affected multiple systems. However, Neu5Gc is found in human cancers and fetuses and in trace amounts in normal human tissues, suggesting an alternate biosynthetic pathway. We inactivated the mouse Cmah gene and studied the in vivo consequences. There was no evidence for an alternate pathway in normal, fetal, or malignant tissue. Rather, null fetuses accumulated Neu5Gc from heterozygous mothers and dietary Neu5Gc was incorporated into oncogene-induced tumors. As with humans, there were accumulation of the precursor N-acetylneuraminic acid and increases in sialic acid O acetylation. Null mice showed other abnormalities reminiscent of the human condition. Adult mice showed a diminished acoustic startle response and required higher acoustic stimuli to increase responses above the baseline level. In this regard, histological abnormalities of the inner ear occurred in older mice, which had impaired hearing. Adult animals also showed delayed skin wound healing. Loss of Neu5Gc in hominid ancestors approximately 2 to 3 million years ago likely had immediate and long-term consequences for human biology.

FIG. 1.

Generation of the human-like Cmah^−/− mouse. The targeting construct and final targeted Cmah allele are shown.

FIG. 2.

Cmah^−/− mice are deficient in Neu5Gc. (A) Neu5Gc expression was studied by immunohistochemistry in multiple tissues from multiple animals using a chicken anti-Neu5Gc antibody, and examples are shown. The few null tissues staining weakly positive were studied by acid hydrolysis, DMB-HPLC, and mass spectrometry analysis, showing no evidence of Neu5Gc. (B) Increased 9-O acetylation of Neu5Ac in Cmah null mice. The CHE-FcD probe was precomplexed with horseradish peroxidase-conjugated goat anti-human IgG at an optimized ratio and applied to sections of tissues from WT and Cmah^−/− animals. Magnification, ×100. Brownish-red color represents positive staining.

FIG. 3.

No alternative pathway for Neu5Gc biosynthesis in fetuses or tumors. Neu5Gc expression was studied by immunohistochemistry as with Fig. 1. (A) Fetuses from Cmah^−/− female mice with or without 1 mg/ml free Neu5Gc in the drinking water showed similar staining patterns with the anti-Neu5Gc antibody, indicating that reactivity is not due to uptake and incorporation. (B) Cmah^−/− MMTV-PyMT transgenic mice were given Neu5Gc (1 mg/ml) free in the drinking water for 1 month after onset of mammary tumor growth. Positive staining for the anti-Neu5Gc antibody was detected, indicating that dietary Neu5Gc can be incorporated into tumors. Magnification, ×100. (C) A Cmah^+/− female mouse was mated to a Cmah^+/− male mouse. Pups were studied immediately after birth for Neu5Gc expression. Immunohistochemistry analysis of Cmah^+/+, Cmah^−/−, and Cmah^+/− fetuses showed strong reactivity to all organs except for the brain.

FIG. 4.

Behavioral differences between Cmah^−/− and WT mice. (A) Startle reflex differences represented by data from the fourth cohort. This result is representative of similar findings in three of four experiments. (B) ABR thresholds. A cohort of older (9 months) Cmah^−/− mice had significantly reduced hearing sensitivity across the frequency range.

FIG. 5.

Histological abnormalities in inner ears. (A) In the vestibular otoconial epithelia, unusual deposits of apparently acellular material were present on the apical surface of the epithelium, among the stereociliary bundles. (B) In the cochlear sensory epithelium, the area of the outer hair cells showed degeneration of the sensory cells throughout the cochlea, with collapse of the outer portion of the organ of Corti in the basal (high-frequency) turn. (C) Fluid-filled cyst observed on a semicircular canal organ of a null animal.

FIG. 6.

Differences in cutaneous wound repair. A single wound was made with a dermal biopsy punch on the dorsal skin and measurements of wound area done by taking daily digital images. Data represent one experiment, with significance by one-way analysis of variance of a P value of <0.0001, seven mice per group, and are representative of similar findings in three experiments.