A mutation in human CMP-sialic acid hydroxylase occurred after the Homo-Pan divergence

Abstract

Sialic acids are important cell-surface molecules of animals in the deuterostome lineage. Although humans do not express easily detectable amounts of N-glycolylneuraminic acid (Neu5Gc, a hydroxylated form of the common sialic acid N-acetylneuraminic acid, Neu5Ac), it is a major component in great ape tissues, except in the brain. This difference correlates with lack of the hydroxylase activity that converts CMP-Neu5Ac to CMP-Neu5Gc. Here we report cloning of human and chimpanzee hydroxylase cDNAs. Although this chimpanzee cDNA is similar to the murine homologue, the human cDNA contains a 92-bp deletion resulting in a frameshift mutation. The isolated human gene also shows evidence for this deletion. Genomic PCR analysis indicates that this deletion does not occur in any of the African great apes. The gene is localized to 6p22-p23 in both humans and great apes, which does not correspond to known chromosomal rearrangements that occurred during hominoid evolution. Thus, the lineage leading to modern humans suffered a mutation sometime after the common ancestor with the chimpanzee and bonobo, potentially affecting recognition by a variety of endogenous and exogenous sialic acid-binding lectins. Also, the expression of Neu5Gc previously reported in human fetuses and tumors as well as the traces detected in some normal adult humans must be mediated by an alternate pathway.

Figure 1

Comparison of nucleotide and derived amino acid sequences of CMP-Neu5Ac hydroxylase cDNAs from mouse, chimpanzee, and human. (A) Comparison of nucleotide sequences. Sequencing of multiple full-length cDNA clones was carried out as described. The ClustalW program (http://www2.ebi.ac.uk/clustalw/) was used to align the sequences. The 92-bp gap in the human cDNA and differences at the 3′ end are indicated with dotted lines. The start and stop codons of the primary ORF are underlined. (B) Comparison of the ORFs of the cDNAs. The putative Reiske iron-sulfur binding region of the mouse and chimpanzee hydroxylase are underlined.

Figure 2

PCR analysis of genomic DNA from humans and African great apes. Genomic DNA from human (Hu), chimpanzee (Ch), bonobo (Bo), or gorilla (Go) was subjected to PCR analysis with primers corresponding to the 5′ and 3′ ends of the 92-bp sequence missing from the human cDNA. S, molecular weight standards; +, positive control, using chimpanzee cDNA as template; −, negative control, without template. Some higher molecular weight background bands seen with great ape DNA were more prominently amplified in the human sample.

Figure 3

Southern blot analysis of a BAC clone encompassing the human hydroxylase gene. BAC 443N17 DNA (10 μg) was digested with BamHI (HI) or EcoRI (RI) and subjected to Southern blot analysis using probes corresponding to the missing region in the human cDNA (PR, based on chimpanzee sequence), and the sequences immediately 5′ (P5) and 3′ (P3) of this region. All three probes were equally sensitive when used to probe Southern blots of the chimpanzee hydroxylase cDNA (data not shown).

Figure 4

Chromosomal localization of the human CMP-sialic acid hydroxylase gene. (A) Human/rodent somatic-cell hybrid mapping. Ethidium bromide-stained agarose gel analyzing the products of PCR amplification of samples of the NIGMS human/rodent somatic-cell hybrid mapping panel 2. Lanes labeled 1–22, X, and Y reflect DNA from a chromosome 6 hybrid clone containing the designated human chromosomes. Hamster (Ha), mouse (Mo), and human (Hu) are total genomic DNA from those species used to create the hybrid panel. A negative control (B) is shown as well as 100-bp marker lanes with the 300- and 400-bp bands indicated. (B) FISH mapping. Metaphase FISH localization of genomic BAC clone 443N17 containing the CMP-sialic acid hydroxylase gene to the distal short arm of human chromosome 6. Detection of digoxigenin-labeled probes was achieved with anti-digoxigenin antibodies conjugated to rhodamine (giving the red signal), followed by 4′,6-diamidino-2′-phenylindole dihydrochloride (DAPI) counterstaining of the chromosomes and viewing under an epifluorescence microscope with a triple band pass filter.