Minimal mutations are required to effect a radical change in function in CEA family members of the Ig superfamily

Abstract

GPI anchorage in the CEA family results in the acquisition of radically different functions relative to TM anchorage, including inhibition of differentiation and anoikis, disruption of tissue architecture and promotion of tumorigenicity. CEA GPI anchors, as determined by the carboxy-terminal exon of CEA, demonstrate biological specificity in their ability to confer these functional changes. CEA family GPI anchorage appears to have evolved twice independently during the primate radiation, in a manner suggestive of evolution from more primitive TM-anchored CEACAM1. We show here that very few mutations in the TM exon of present-day human CEACAM1 are required to give efficient GPI anchorage and the biological specificity of CEA GPI anchors, i.e., to give the differentiation-blocking function of GPI-anchored CEA. Such a change in anchorage could therefore represent a relatively facile means for producing radical change in molecular function of Ig superfamily members during evolution.

Evolution of a Tumorigenic Property Conferred by Glycophosphatidyl-Inositol Membrane Anchors of Carcinoembryonic Antigen Gene Family Members during the Primate Radiation

“…On the assumption that a stop codon in the carboxy-terminal domain of CEA family members is indicative of GPI linkage (Naghibalhossaini and Stanners, 2004), the carboxy-terminal exon nucleotide sequence of human CEA was used to design two sets of primers for PCR reactions with genomic DNA of various species (Figure 1). One of these sets (S-1 and AS-1) could amplify only sequences containing the human CEAlike stop codon, because the 3Ј end of the AS-1 primer is situated in the stop codon.…”

“…We have shown that incomplete GPI-processing of CEACAM1 proteins with mutant anchor-determining domains could be distinguished by the presence of a less glycosylated lower-molecular weight, Triton X-100-soluble band that is localized intracellularly (Naghibalhossaini and Stanners, 2004). Because no such band could be seen in the immunoblot of the CC1-CMO chimeric protein ( Figure 4B), we conclude that this chimeric protein, as with all of the naturally occurring GPI-linked human CEA family members (Naghibalhossaini and Stanners, 2004), is efficiently GPI processed. Evolution of key mutations in the anchor-determining exons of CEA gene family members giving GPI anchorage relative to the phylogenetic tree of various primate and related species (Porter et al, 1997).…”

“…We previously showed that several GPI-anchored CEA family members and even a GPI-anchored construct derived from TM-anchored CEACAM1 could block myogenic differentiation of L6 myoblasts (Rojas et al, 1996;Naghibalhossaini and Stanners, 2004). Because this finding represents an opportunity to gain insight into the normal functions of the GPI-linked members of the CEA family, and considering the evidence that GPI anchors derived from the expression of different carboxy-terminal exons can determine specificity of function (see Introduction), it was important to investigate whether the novel GPI anchor confers functions similar to those of the common human CEA GPI anchor.…”

“…We have shown recently that such mutation can be achieved relatively easily: the introduction of a stop codon alone into the TM-determining domain of human CEACAM1 converted this CEA family member into a GPI-anchored protein, albeit one that was inefficiently processed. Two further mutations upstream of the stop codon conferred the 100% processing efficiency seen in naturally occurring GPI-anchored CEA family members (Naghibalhossaini and Stanners, 2004). These considerations therefore raise an important question: can a structural feature that changes the function of CEA family members toward the perturbation of cell and tissue architecture and the inhibition of differentiation be selectively advantageous and therefore maintained in evolution?…”

“…Both the sequences with the CEA package and the Ceb package possess, in addition to the stop codons, upstream changes (relative to the sequences of TM-anchored domains) necessary for efficient GPI processing (Englund, 1993). The latter (Figure 3, open arrowheads) represent changes toward hydrophilicity, one of which, the I-to-T change (first bolded amino acid in consensus sequences; also underlined in Figure 1 as the ACT codon), has been shown directly to markedly enhance the GPI processing efficiency of CEACAM1 mutated to include the CEA stop (from 5 to 56%) (Naghibalhossaini and Stanners, 2004). The "consensus" sequences obtained from all species for five such residues (indicated in bold in Figure 3), including the abovementioned T residue, are strikingly similar for both the CEAstop and Ceb-stop GPI domains, and they differ from these residues in the consensus sequence of TM domains.…”