A total of 62 CD300A and CD300C sequences, also referred to as CMRF35-like molecules 8 and 6 respectively, from 33 primate species were retrieved from the National Center for Biotechnology Information (NCBI) GenBank database. Sequence retrieval was initiated with BLAST searches using the human CD300A (NM_007261) and CD300C (NM_006678) reference sequences as queries. Subsequently, additional BLAST searches were performed using representative sequences from major primate groups to identify orthologs that may not have appeared in the initial query. Additionally, CD300 sequences from Tupaia chinensis were included as outgroups to provide a broader evolutionary perspective.

Synteny analysis was performed using the NCBI Genome Data Viewer to investigate the genomic organization of the CD300 genes. Orthologs were further validated by conducting BLAST searches in public databases (NCBI BLASTn) comparing the sequences across species to confirm homology. Genes adjacent to the CD300 gene cluster (GPRC5C and RAB37) were used as genomic anchors to assess the conservation of synteny.

Sequence alignments were performed using the ClustalW Multiple Alignment tool implemented in the BioEdit Sequence Alignment Editor. Manual adjustments were applied to maintain correct reading frames and improve alignment quality, particularly in regions with potential frameshifts or indels.

Phylogenetic analyses were performed in MEGA X (version 11.0.13) using the maximum likelihood (ML) method. The amino acid phylogenetic tree was constructed based on the highly homologous portion of the extracellular domain (around 112 amino acids) of CD300A and CD300C, representing the most conserved region among CD300 genes. The optimal nucleotide substitution model for the dataset was determined in MEGA X to be JTT+G with five gamma-distributed rate categories. Bootstrap analysis with 1000 replicates was conducted to evaluate the trees’ robustness.

To assess amino acid variability, separate multiple sequence alignments were conducted for each gene using their complete coding sequences. These alignments allowed the identification and analysis of conserved cysteine residues (involved in disulfide bond formation), glycosylation sites, and potential pseudogenization events (premature stop codons or frameshift mutations). Sequence variability was analyzed across primate families to identify evolutionary patterns potentially linked to ecological adaptations or selective pressures.

The conservation of ITIM sequences in CD300A and the charged glutamic acid residue in CD300C was also evaluated. These analyses aimed to determine the degree of conservation of the ITIM consensus sequence and the critical transmembrane residue in CD300C, identifying sequence variations that could alter, disrupt, or abolish receptor signaling and ultimately affect their immune regulatory function. The human CD300A and CD300C three-dimensional models ( Figure 2A ) were obtained with Swiss model using the sequences from Uniprot (accession numbers: Q9UGN4 and Q08708 respectively).

As noted previously ( Table 1 ), several primate species exhibit clear signs of CD300C pseudogenization or complete gene loss, contrasting sharply with the strong evolutionary conservation of CD300A. A particularly interesting case is the Hylobatidae family, in which the entire CD300 gene complex exhibits a unique and highly rearranged genomic organization when compared to other primates ( Figure 3 ). Multiple CD300 genes, including CD300C, are missing, and others have relocated to different genomic regions, no longer flanked by the typical syntenic markers GPRC5C and RAB37. This pattern strongly suggests that a major chromosomal rearrangement occurred in the common ancestor of Hylobatidae, approximately 9 million years ago (17), leading to the disruption of the original CD300 locus and the subsequent loss of CD300C and other CD300 genes, such as CD300LD.

Genomic rearrangements such as deletions, translocations, and inversions can significantly impact gene families by altering their regulatory landscapes, potentially affecting their expression levels or functional interactions. However, the persistence of this rearrangement across all analyzed Hylobatidae species suggests that it was not a detrimental event but rather one that was either neutral or conferred a selective advantage, indicating that this species did not rely heavily on these receptors or that an alternative immune pathway evolved to have similar functions.

A comparable but independent pattern of CD300C loss is observed in Lemur catta, which also lacks CD300C, CD300LD, CD300H, and CD300E. However, unlike Hylobatidae, L. catta retains the typical CD300 genomic region, with CD300 genes flanked by GPRC5C and RAB37, indicating that the loss occurred without major chromosomal rearrangements. This reinforces the hypothesis that CD300C loss resulted from an independent event. However, as in Hylobatidae, only activating receptors were lost, while inhibitory members of the CD300 family were preserved. The selective loss of activating receptors such as CD300C in both L. catta and Hylobatidae suggests that immune systems in these species may have favored reduced or tightly regulated activation. This may reflect adaptation to a lower pathogen burden, a more stable ecological niche, or the need to prevent immunopathology through hyperactivation. It is also possible that the relocation of some retained CD300 genes in Hylobatidae could allow for new regulatory interactions, placing them under the influence of different promoters, enhancers, or chromatin environments. If these changes resulted in a more efficient immune strategy, conferring greater resistance to pathogens or a reduced risk of immune-mediated tissue damage, they would have been positively selected over evolutionary time.

The evolution of the CD300 gene family in primates follows the birth-and-death molecular evolution. This type of dynamic gene evolution is not unique to the CD300 family. Numerous immune gene families, such as the major histocompatibility complex (MHC) (15, 25), have undergone lineage-specific duplications, losses, and pseudogenization in primates and other mammals. Similar patterns have also been reported for gene families in primates concerned with innate immunity, such as killer cell immunoglobulin-like receptor (KIR) (15, 26), guanylate binding proteins (GBP) (27), tripartite motif (TRIM) (28), and interferon-inducible transmembrane proteins (IFITMs) (29). These families are well-established examples of multigene families evolving under the birth-and-death model. Such an evolutionary pattern is common among immune-related multigene families because it reflects the intense selective pressures exerted by the pathogen-host arms race. In this dynamic conflict, pathogens rapidly mutate to evade immune detection, while hosts counteract through gene diversification via duplications, positive selection, and the elimination or pseudogenization of obsolete variants.

Previous studies have demonstrated that all four ITIMs contribute to CD300A’s inhibitory function. However, the third ITIM (Y267) is the most important one. Its mutation leads to the loss of most of its ability to inhibit immune responses. Moreover, when the third ITIM is intact, the functional impact of mutations in two of the other ITIMs is limited and doesn’t significantly affect the inhibition of CD300A (11). To evaluate the evolutionary conservation of CD300A ITIMs ( Figure 2A ), sequence alignments were performed across all analyzed primate species ( Figure 2B ). The first three ITIMs follow the canonical consensus motif for classical ITIMs (I/V/L/SxYxxL/V). In the first ITIM (residues 229-234), tyrosine (Y) is conserved across all species, although the initiating residue (I/V/L/S) is replaced by glutamine (Q) and glutamic acid (E) in some species. The second ITIM is the most conserved one, being completely in agreement with the classical ITIM consensus sequence in all the species.

In the third ITIM, three species exhibit a substitution of the initiating residue (I/V/L/S) with proline (P). However, the tyrosine is conserved, and the remaining sequence aligns with the classical ITIM consensus sequence.

The fourth ITIM follows the non-classical consensus motif (I/V/L/S/TxYxxL/V/I). However, this ITIM also shows variability, especially in Lemuriformes and Lorisiformes (Microcebus murinus, Propithecus coquereli, Nycticebus coucang, Lemur catta, and Otolemur garnettii), where the initial residues (I/V/L/S/Tx) are missing, although the tyrosine itself is preserved. Whether these variations impair inhibitory signaling remains unclear and warrants further functional validation.

Since CD300C possesses a short cytoplasmic tail lacking ITAMs, its activating signal relies on a charged glutamic acid residue (E191) within the transmembrane domain, which mediates interaction with ITAM-bearing adaptor proteins ( Figure 4A ). Sequence alignments across primate species revealed that the glutamic acid (E191) was replaced by lysine (K) in seven species: three from the Cebidae family (Callithrix jacchus, Aotus nancymaae, and Saimiri boliviensis boliviensis) and four others (Microcebus murinus, Propithecus coquereli, Nycticebus coucang, Otolemur garnettii) ( Figure 4B ). This substitution may disrupt interaction with adaptor proteins, leading to non-functional or impaired signaling capacity. In principle, lysine (positively charged) could mediate interactions with alternative adaptor proteins that carry complementary negative charge. However, the replacement of the negatively charged glutamic acid with a positively charged lysine may interfere with the electrostatic interactions with the adaptor protein. Activating receptors, such as CD300C, need to associate with specialized signal-transducing transmembrane polypeptides due to the absence of intrinsic signal motifs. This association occurs within the transmembrane domain and depends on the formation of non-covalent interactions between oppositely charged amino acid residues in the receptor and the adaptor molecule (10). Studies have shown that mutating this glutamic acid to a valine (E191V) abolishes signaling through FcRγ, even though the receptor still physically interacts with the adaptor. This suggests that the negative charge of glutamic acid plays a critical role in signal transduction. The study does not directly clarify whether the signaling failure of the E191V mutant is solely due to the loss of negative charge or also due to the introduction of the hydrophobic valine residue. However, the available evidence suggests that the key issue is the loss of the negative charge, as the mutation impairs function despite the structural association with FcRγ being preserved. Therefore, it is plausible to theorize that a substitution by lysine, which introduces a positive charge, may affect the activating signal. However, to determine whether this substitution truly affects receptor function, dedicated functional investigations will be necessary (10). Additionally, the CD300C sequence retrieved from Papio anubis was found to be significantly shorter than typical CD300 sequences, lacking both the transmembrane and cytoplasmic domains. This structural deficiency strongly suggests that CD300C in P. anubis is non-functional. However, it was not classified as a pseudogene as it did not meet our predefined criteria for pseudogenization, which included the presence of premature stop codons or frameshift mutations. Together, these patterns reinforce the conclusion that CD300C has undergone multiple independent losses or functional impairments across the primate lineage, through both complete gene deletion (as in Hylobatidae and Lemur catta) and potential functional loss caused by mutations. This supports a birth-and-death model of evolution, where multigene families expand through gene duplication, but some duplicated genes accumulate deleterious mutations, become pseudogenes, or are eventually lost.

A comparative analysis of the highly homologous portion of the extracellular domain sequences of CD300A and CD300C (around 112 amino acids) revealed strong evidence of gene conversion across several primate lineages ( Figure 5 ). In many species, including humans, both receptors exhibit high sequence similarity in this region, displaying a pattern of concerted evolution, in which sequences from related genes are homogenized through gene conversion, likely preserving the ligand-binding site.

This phenomenon is reflected in the phylogenetic tree ( Supplementary Image 1 ), where CD300A and CD300C sequences tend to cluster by species or family rather than by gene type, as typically would be expected. For example, genes from Piliocolobus tephrosceles cluster together regardless of receptor type, suggesting that intra-lineage similarity between both sequences in this region exceeds the inter-lineage similarity among orthologs. This suggests that CD300A and CD300C extracellular domains sequence homogenization has occurred preferentially within lineages, rather than maintaining strict divergence between gene copies. Furthermore, the extent of gene conversion between CD300A and CD300C appears to vary across different evolutionary lineages.

This pattern of gene conversion observed in the extracellular domain between CD300A and CD300C is further supported by the presence of unique shared motifs within certain primate families and species ( Figure 5 ). For instance, the motif 30WYEEKHK36 is exclusive to some Cercophitidae species and is consistently found in both CD300A and CD300C sequences. Similarly, a conserved aspartic acid 93D residue is present in both genes across all representatives of the Cebidae family, reinforcing the idea of concerted evolution within lineages. This phenomenon also occurs at the species level, for example, in Aotus nancymaae, both receptors sequences share the 28HCEYEEK34 motif, while in Macaca nemestrina, the 107HK—DPIVQVQVEMS122 motif is present in both genes, including a three-amino-acid deletion that appears in both sequences. These examples, among many others ( Figure 5 ), highlight how gene conversion events maintain sequence similarity between CD300A and CD300C at different taxonomic levels, reinforcing conservation of the ligand-binding region while allowing divergence in the cytoplasmic tail, maintaining their paired receptor dynamic. However, in species where CD300C has been pseudogenized, this pattern of concerted evolution is disrupted. This disruption across multiple independent lineages suggests that the loss of CD300C is not a shared ancestral trait, but rather a repeated outcome of convergent evolution, where similar selective pressures may have driven the parallel inactivation or elimination of this activating receptor.

The functional constraints and structural features that characterize the CD300A and CD300C genes, essential for receptor stability, are mostly preserved. Cysteine residues (C36↔C103 for CD300A or C43↔C110 for CD300C) involved in disulfide bond formation, which are highlighted in green boxes in Figure 5 , are highly conserved across most primate species. One notable exception is Nycticebus coucang CD300A, where a phenylalanine replaces the cysteine (C→F). The second disulfide bond, corresponding to C57↔C65 in CD300C (blue boxes in Figure 5 ), is also broadly conserved in almost all primates, but is lost in Lemuriformes and Lorisiformes, suggesting lineage-specific alterations that may impact protein stability or ligand-binding efficiency.

Conversely, N-glycosylation sites, highlighted in pink in Figure 5 , display more variability. Although several glycosylation motifs are retained across families, others are altered in certain lineages and replaced by other amino acids. The most frequent substitution observed is asparagine to serine (N→S), occurring in either CD300A, CD300C, or both. For instance, Piliocolobus tephrosceles exhibits shifts in glycosylation sites in both receptors, which may result from gene conversion or adaptive pressures that alter glycosylation patterns. These modifications are particularly evident in some Cercopithecidae, Lemuriformes, and Lorisiformes species, and could influence receptor surface structure, stability, or ligand-binding dynamics.