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Recent evolution of the salivary mucin MUC7

  • Scientific Reports 6, Article number: 31791 (2016)
  • doi:10.1038/srep31791
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Abstract

Genomic structural variants constitute the majority of variable base pairs in primate genomes and affect gene function in multiple ways. While whole gene duplications and deletions are relatively well-studied, the biology of subexonic (i.e., within coding exon sequences), copy number variation remains elusive. The salivary MUC7 gene provides an opportunity for studying such variation, as it harbors copy number variable subexonic repeat sequences that encode for densely O-glycosylated domains (PTS-repeats) with microbe-binding properties. To understand the evolution of this gene, we analyzed mammalian and primate genomes within a comparative framework. Our analyses revealed that (i) MUC7 has emerged in the placental mammal ancestor and rapidly gained multiple sites for O-glycosylation; (ii) MUC7 has retained its extracellular activity in saliva in placental mammals; (iii) the anti-fungal domain of the protein was remodified under positive selection in the primate lineage; and (iv) MUC7 PTS-repeats have evolved recurrently and under adaptive constraints. Our results establish MUC7 as a major player in salivary adaptation, likely as a response to diverse pathogenic exposure in primates. On a broader scale, our study highlights variable subexonic repeats as a primary source for modular evolutionary innovation that lead to rapid functional adaptation.

Introduction

Duplications, deletions, inversions and translocations of genetic segments, collectively called genomic structural variants, constitute an evolutionarily important form of genetic variants1. When two genomes from the same primate species are compared with each other, structural variants constitute 2 to 7 times more base pair differences than single nucleotide variants2. A functionally relevant, but understudied subset of structural variants, is copy number variation of tandem repeats within individual coding exons (subexonic repeat variation)3. A recent study has shown that, despite the high intrinsic mutability of subexonic repeats, the vast majority of thousands of subexonic repeats remain strongly conserved among mammals4. However, there is no study to our knowledge that specifically investigated the evolution of copy number variable subexonic repeats from their initial emergence as functional units to the adaptive constraints and mutational properties that contributed to their extant variation in primates.

Here, we focus on the salivary mucin-7 gene (MUC7) to study the evolution and functional impact of subexonic repeats. MUC7 comprises a number of interesting properties that could make it a model for studying subexonic repeats (Fig. 1A). First, MUC7 carries subexonic repeats that are 69 bp (23 amino acids) long and are copy number variable in humans5. These repeats can be particularly well studied in this gene because the repeats are relatively short and few in number compared to other mucins. Second, the subexonic repeats of MUC7 code for densely O-glycosylated proline-, threonine-, and serine-rich (PTS) tandem repeat domains. PTS-repeats are a hallmark of the mucin group of proteins6. Mucins are highly glycosylated proteins that serve a variety of roles in organisms from lubrication to cell-signaling to defense7. In addition, the dense arrays of O-glycans also create large protease-resistant regions that provide binding sites for commensal microbes and pathogens, thus preventing them from binding epithelial cells8,9. Third, MUC7 codes for an abundant salivary protein10. Recent studies have reviewed the importance of structural variants affecting salivary proteins as a response to rapid changes in human diet and composition of oral exposure to different pathogens1,11. MUC7 provides an opportunity to assess the broader implications of subexonic repeat variation within the context of the functional evolution of the mucin functional group and primate salivary adaptation.

Figure 1: The structure and function of MUC7.
Figure 1

(a) Genomic location, organization and subexonic copy number variation of the MUC7 gene in the human reference genome (GRCh37/hg19). The gray bar indicates the chromosomal band (q13.3) where MUC7 resides. The zoomed-in version of the gene shows the two coding exons (thick blue bars), the UTR region at the 3′ and 5′ ends (thin blue bars) and intronic regions (blue line in-between exons). Start and end of the transcript are indicated by the numbers at each end. The different functional domains within the MUC7 protein are designated by different colors at the bottom of the panel66; (b) Presence or absence of PTS-repeats and of human exon 2 (i.e., first coding exon coding for the conserved signal peptide with cysteine residues detectable by BLAST search). The presence of significantly similar sequences (E-value < 0.01) to human exon 2 is indicated with red and the absence with gray. The polar bear MUC7 ortholog contains a truncated version of exon 2 with only 36 of 54 bp represented (shown in hatched pattern). The presence of PTS repeats is shown in blue. The absence is shown in grey. The phylogenetic tree on the right indicates the species relationships between the organisms analyzed here; (c) Percentage of T and S amino acids (indicative of potential O-glycosylation sites) in relation to the total number of amino acids for MUC7 orthologs and other proteins belonging to the SCPP gene family across mammals. MUC7 shows a significantly higher TS percentage compared to other salivary proteins in SCPP gene family (based on the NCBI gene model; P-value between MUC7 and PROL1 is 1.123 × 10−09 by one-tailed student’s t-test).

MUC7 belongs to the secretory calcium-binding phosphoprotein (SCPP) family, which likely evolved after the divergence of bony and cartilaginous fish12. The SCPP gene family resides at the short arm of chromosome 4 in humans. It also includes other salivary proteins, such as histatins, proline-rich proteins, and statherin, as well as non-salivary proteins, such as milk caseins and enamel matrix proteins. In addition to the PTS-repeats, the MUC7 protein contains a non-glycosylated “naked” region at the N-terminus of the apomucin molecule that shares homology with salivary histatins. Similar to them, this region exhibits bactericidal and fungicidal properties13. MUC7 also contains two moderately N- and O-glycosylated domains flanking the PTS repeat domain, and a C-terminal domain that contains a leucine-zipper motif14. Functionally, the comparatively small non-gel-forming MUC7 contributes relatively more to the innate immune functions of saliva than to the lubricating or barrier-forming properties typical for the much larger gel-forming salivary mucins, such as MUC5B and MUC1915,16.

Even though MUC7 is grouped with other mucin proteins by virtue of their biochemical and functional similarities, it shares no sequence similarity or known recent common evolutionary ancestor with any of the other known mucin genes6. Other than its syntenic relationship with the SCPP gene family and the functional similarity to other mucins, the evolution of MUC7 remains unclear. Several attempts were made to pinpoint the origins of different mucins and follow their evolutionary trajectories6,17,18,19,20. The defining feature of mucins, PTS-repeats, have been reported in MUC7 to range from 5 to 6 haploid copies in humans21. Functionally, the less-common 5-copy allele was suggested to be associated with protection against asthma5,22. From an evolutionary standpoint, the copy number variation of MUC7 PTS repeats in nonhuman primates have not been fully explored. Therefore, in this study, we conducted genomic analyses to elucidate the evolution of MUC7 in primates, especially with regards to its subexonic repeats.

Results

MUC7 likely originated in the placental mammal ancestor as a functional gene

MUC7 is evolutionarily related to SCPP gene family through gene duplication23. The evidences for this are the syntenic location of MUC7 within the SCPP gene cluster and the presence of a signal peptide, which is a functionally fundamental hallmark of all extracellular matrix proteins24. However, it is not clear when MUC7 emerged as a functional gene. Based on current gene prediction models, it appears that MUC7 orthologs are restricted to placental mammals25. However, gene predictions, especially for genes that have repeat content as in MUC7, may be error prone26. To independently investigate the presence or absence of MUC7 orthologs, we used BLAST27 to identify similar sequences to the non-copy number variable first coding exon (second exon, which codes for the signal peptide) of human MUC7 in multiple vertebrate reference genomes (Figure S1). Our results are concordant with the gene models where MUC7 is present in most placental mammals, but absent in marsupials (Fig. 1B). Although MUC7 orthologs could not be found in rat and mouse reference genomes, it was recently reported that rodents express a salivary mucin gene that functionally resembles MUC715. It is therefore possible that this gene has evolved convergently, filling the adaptive niche for a salivary-specific small mucin in rodents after lineage specific deletion of MUC7. There are some caveats in our analyses. First, It is plausible that lineages such as marsupials, where we could not detect MUC7-like sequences, originally carried the MUC7 gene but may have lost it during their evolution, similar to rodents. Second, it is also plausible that the signal peptide sequence, that we used for BLAST analysis, may have diverged in some lineages. Thus, starting from the primate MUC7 signal peptide sequence, we may not have had the statistical power to detect such diverged sequences in some species. In fact, when we repeated the BLAST analyses using the MUC7 histatin-like sequence instead, we could detect significantly similar sequences only in primates, even though syntenic histatin-like sequences are present in most mammalian MUC7 genes (Figure S1). This exemplifies the shortcomings of BLAST analysis for definitively pinpointing to the origin of the MUC7 gene. Having stated the caveats, still the most parsimonious explanation, based on the analyses presented here, is that MUC7 evolved in the placental mammal ancestor.

The ancestral MUC7 gene has rapidly gained PTS repeats after its emergence

One question is when did MUC7 gain heavy O-glycosylation through its PTS-repeats. The clusters of T and S amino acids in proteins were discussed in multiple previous studies as targets for O-glycosylation14,28. In fact, mucins, as a functional group, are defined by the presence of PTS-rich repeats6, where T and S clusters provide targets for O-glycosylation. For human MUC7, the heavy glycosylation of the PTS-repeats has been demonstrated experimentally29. Therefore, we first asked whether PTS-rich repeats exist in all MUC7 non-human orthologs. To address this question, we conducted multiple overlapping analyses to minimize biases due to the variation in the quality, completeness and annotation of reference genomes. First, we used BLAST to identify sequences across available mammalian genomes that are similar to the PTS-repeats observed in humans (E-value < 0.01). Second, we investigated the presence or absence of subexonic repeats across mammalian reference genomes as annotated by Simple Tandem Repeat track30 as well as through manual inspection within the predicted MUC7 coding sequences. Collectively, our analysis showed that MUC7 coding sequences in most placental mammals comprise repeated PTS-sequences (Fig. 1B).

Certain mammalian species, such as dog, cow, horse, camel and shrew also showed no evidence for PTS-repeat sequences. This may either indicate lineage-specific loss of repeats, polymorphism within species, or an artifact resulting from reference genome assembly errors31,32. It should be noted here that most other mucins, including MUC1, MUC4, and MUC6, have intact PTS-repeats in mouse33,34,35, although the sequences of these repeats can vary between humans and mice33. As such, a complete loss of all PTS-repeats of a mucin gene is probably a rare event in evolution. Thus, the observed lack of MUC7 PTS-repeats in at least some of the mammalian species mentioned above could well be due to reference genome errors. Indeed, similar incorrect representations of tandem duplications in reference genomes have been reported earlier36.

To evaluate whether the PTS-repeats in other mammalian species are defined, as in humans, with high proportion of T and S amino acids, we measured the number of codons encoding for T and S amino acids in the coding sequences of MUC7 genes in extant mammals (Fig. 1C). We found that, regardless of the number of repeats found in the gene, all MUC7-coding sequences in mammals have a proportionally higher T and S content than other members of the evolutionarily related SCPP gene family (e.g., MUC7 T and S percentage is significantly higher than the adjacent PROL1 among mammals, P-value = 1.123 × 10−9, one-tailed Student’s t-test). We also compared the proportion of T and S amino acids between MUC7 and another salivary mucin gene, MUC5B. We found that the proportion of T and S amino acids in MUC7 is also consistently higher than MUC5B among mammals (Figure S2, P-value = 0.004382, one-tailed Student’s t-test). These results suggest that the ancestral MUC7 likely gained TS rich, PTS-repeats early after its emergence.

The chromosomal location of MUC7 likely determined saliva-specific expression

MUC7 is expressed primarily in saliva, and several other genes in the SCPP gene family are also expressed in saliva12. Once a gene duplication happens, such as the one which led to initial genesis of MUC7, it is plausible that the cis-regulatory sequences of the ancestral gene are also copied, thus regulating the expression of the duplicated gene. Indeed, recent studies have shown that tandemly duplicated genes have an increased chance of having similar expression profiles2,37. To test whether the saliva-specificity of MUC7 expression is similar to the neighboring members of the SCPP family, we used a global gene-expression database, GTEx38 (Table S1). Briefly, we compared the expression in minor salivary gland (the only salivary gland presented in GTEx dataset) to the expression in all other GTEx tissues. Then, we calculated a nominal P-value for saliva-specific expression for all genes across the genome. This analysis showed there is an unexpectedly high number of genes with saliva-specific expression adjacent to MUC7 when compared to the rest of the genome (Wilcoxon rank-sum test, nominal P-value = 2.2 × 10−16, Fig. 2A,B). Thus, it is plausible that the gene cluster where MUC7 emerged has had saliva specific expression for a long evolutionary time and that the location of its emergence predetermined the expression pattern of of MUC7.

Figure 2: The MUC7 gene is located within a chromosomal region where most of the genes show saliva-specific expression.
Figure 2

(a) We searched chromosome 4 for 15-gene windows (i.e., each window is defined by 15 adjacent genes). Then, for each gene we calculated the likelihood of saliva-specific expression by comparing the expression of this gene in minor salivary glands as compared to all other non-salivary tissues in the GTEx dataset using a Wilcoxon rank-sum test (See methods for the details). The x-axis shows the number of genes in a given window that are expressed primarily in saliva (i.e. expression in minor salivary gland is significantly higher than in other tissues (P-value < 0.01)). MUC7 is located in the window with the highest number of genes having salivary gland-specific expression (10 genes). The y-axis shows the frequency of observed windows in squared-scale (Wilcoxon rank-sum test, P-value = 2.2 × 10−16); (b) This plot shows the likelihood of saliva-specific expression for the chromosomal region that MUC7 resides on chromosome 4. In the scatter plot, the y-axis is the P-value (same way the P-value is calculated for the histogram in Fig. 2A) for saliva-specific expression. The x-axis is the chromosomal location on chromosome 4. The gray dots indicate the genes flanking the “window” in which MUC7 resides, while red dots indicate genes in the same window. The cartoon above the scatter plot is a zoomed-in depiction of the location of MUC7 and the SCPP cluster of genes that show high saliva expression.

The MUC7 PTS-repeats exhibit very high and recurrent copy number variation in mammals and primates, but remain T and S rich

To better understand the evolution of PTS-repeat copy number, we tested whether the PTS-repeat copy number between-species variation is unusual as compared to other subexonic repeats across the genome. To do this, we used a recently available database4, which characterized subexonic repeat content in dozens of different eukaryotic genomes. According to their analysis, MUC7 PTS-repeats were in the upper 0.4% and 17% for subexonic repeat cross-species divergence in nucleotide and copy number divergence, respectively (Figure S3).

To further understand the evolution of the between-species divergence, we tested whether recurrent evolution of PTS-repeats explains some of the observation among primates. To do this, we constructed a heatmap based on the pairwise difference matrix between individual repeat sequences available in reference genomes among primate species (Fig. 3A). We observed that all of the repeat sequences in rhesus macaques cluster with each other, indicating a burst of independent duplication events in the rhesus macaque lineage. Similarly, 3 out of 7 repeat sequences in the orangutan cluster with each other, indicating an expansion of these repeats within the orangutan lineage. We also confirmed these observations using a strongly supported phylogenetic tree based on individual repeat sequences in great apes (Figure S4), as well as using only synonymous SNPs (Figure S5) to ensure that our observations are not due to functional convergence. PTS-repeats in the analyzed species remain in-frame, 69 coding nucleotides in all the species observed, reducing the potential alignment errors. Overall, our results suggest that some of the individual repeats evolved recurrently through lineage-specific duplication events in different primate species.

Figure 3: Presence of MUC7 gene and PTS tandem repeats in mammals.
Figure 3

(a) Heatmap of pairwise nucleotide differences between each repeat sequence within and among primate MUC7 PTS-repeats. Different colors on the right and bottom axes indicate different species. The number shows the position of those repeats in their MUC7 repetitive regions (e.g., Rh_1 indicates the first repeat from the 5′ in the rhesus macaque reference genome). The colors in the heatmap show the nucleotide differences between each pair of repeats, with warmer colors indicating a higher number of nucleotide differences. The groupings (clusters) shown on top and to the left of the heatmap were constructed based on sequence similarity without any a priori hypothesis. Note that, if there is no recurrence and most repeats share a common ancestor, then we expect to see clustering of orthologous repeats. Instead, we observed clustering of repeats within species, indicating species-specific duplication events; (b) Number of total T and S amino acids in each MUC7 protein in relation to the number of TS tandem repeats in primates and other mammals. Relevant species and apparent outliers were indicated by a red circle and their names on the graph. If a species did not show any subexonic repeat content, it is designated by 1 on the x-axis; (c) For each repeat in primate species, the number of pairwise amino acid changes is strongly correlated with pairwise nucleotide changes both across species and within species (R2 = 0.8643), while (d) the number of pairwise TS amino acid changes are not (R2 = 0.03266). The numbers of pairwise TS amino acid remain similar within and among primate species.

To investigate the effect of PTS-repeat copy number on potential O-glycosylation sites within the MUC7 protein, we plotted the number of repeats in mammalian species over the proportion of T and S amino acids in these proteins (Fig. 3B, Table S2). We found them to be highly correlated for both non-primate mammals (R2 = 0.5058) and primates (R2 = 0.5508). If TS-dependent O-glycosylation of these repeats does not have a major fitness effect (e.g., under neutral evolution), mutations should accumulate and revert to a lower percentage of clustering of T and S content than expected by chance. Indeed, the number of nonsynonymous differences from the pairwise comparison of individual repeats within and between species strongly correlate with the number of synonymous differences (R2 = 0.8643, Fig. 3C). However, for T and S amino acids the numbers remain similar, independent of overall nucleotide differences of individual repeats from each other (R2 = 0.03266, Fig. 3D). This supports the notion that TS-dependent O-glycosylation is the main feature that is under adaptive constraints.

In addition to the above-described analysis, we considered that the enzymes, namely the GalNAc transferases, confer O-glycosylation to the polypeptide backbone in a site-specific manner39. This implies that in addition to the number of T and S amino acids, their relative positioning and adjacent amino-acids determine the overall glycosylation potential of the protein. Specifically for MUC7, GalNAc transferases are known to target PTS sequences in such a site specific manner39. To test to what extent these recognition motifs are conserved, we aligned all of the primate mucin repeats to each other. This analysis revealed that the GalNAc transferase recognition motifs as they were defined in humans39, are clearly retained among primates (Figure S6). Overall, our findings show that both the number of T and S amino acids as well as their context within individual PTS repeat domains remain highly conserved. This points to O-glycosylation as the primary driver of adaptive evolution within MUC7 PTS repeats.

Copy number variation of MUC7 PTS repeats has likely evolved under adaptive constraints

To further understand the adaptive forces that have shaped MUC7 PTS-repeat copy number variation in primates, we documented copy number variation of MUC7 PTS-repeats within and across primates, using polymerase chain reaction (PCR)-based genotyping across 10 non-human primate species (Fig. 4A, Table S3). Our results revealed within-species copy number variation for the PTS-repeats (Fig. 4B) where 3 out of 4 great ape species showed common subexonic copy number variation in this gene. We extended our analysis to Old World monkeys, and despite the very limited number of samples we surveyed, we observed copy number variation of PTS repeats. Note that the Old World monkey reference genomes consistently harbor lower numbers of mucin PTS-repeats than we observed by PCR-based assays in non-human primates. This may be due to either uncaptured variation in our sample or misassembly in the reference genomes. Regardless, MUC7 PTS-repeats show high levels of intra-species copy-number variability in the majority of primate species. In general, within-species exonic copy number variation is rare and recurrent, within-species variation in multiple species is even rarer. Such variants have often associated with adaptive, and on occasion, diversifying selection40,41,42,43,44. To sum these results, it is clear that the mutation rate (i.e., the rate of gain and loss mutations) is high for MUC7 PTS-repeats, contributing to within-species copy number variation. However, the range of copy number variation (i.e., 4 to 7 in great apes) is relatively low considering the high mutation rate and may have been shaped with species-specific adaptation. Therefore, we hypothesized that the copy number of PTS-repeats evolves under a fast mutation rate, but was confined to an adaptive range in each individual species.

Figure 4: Copy number variation of MUC7 PTS-repeats in primates.
Figure 4

(a) Copy number variation of MUC7 PTS-repeats genotyped by PCR (Table S3); and (b) compared to the reference genome copy number as indicated by asterisks. The black dots and dashed lines indicate the range of observed PTS repeat copy numbers; (c) Heatmap depicting the comparison of simulated (Table S4) and observed (Table S3) PTS-repeat copy number variation. The x-axis shows the mutation rates that we used for simulations (e.g., θ = 1 corresponds to 1 duplication/deletion event per million years). For each cell in the heatmap, we calculated the ratio between the expected and observed copy number variation. The red colors indicate higher observed variation as compared to simulated variation, whereas the white colors indicate the opposite. The dendrogram to the left is constructed without any a priori input by hierarchical clustering of the values in individual cells in each row. The row labels on the right indicate within species comparisons (green) and among species comparisons (gray). There is consistently more-than-expected variation among great apes and old-world monkey species (upper rows) than within-species and within-families (lower rows). The within-species variation in baboon does not fit in this general trend, possibly because of ascertainment bias due to small sampling.

To test our hypothesis, we simulated the expected range of copy number within and across great-ape species under neutral expectations and under various mutation rates of copy gain/loss and different ancestral copy-number states (Table S4). Since we saw recurrent copy number gain and loss independently within humans, gorillas and orangutans, we surmised that at least one gain or loss has happened in each of these lineages. The average coalescence time for these species are not more than 500,000 years ago and, for humans much less45. Since we observed copy number variation within orangutans, gorillas and humans independently, we can assume a conservative mutation rate of at least one copy number gain or loss every 500,000 years. Therefore, we considered mutation rate scenarios ranging from 0.5 to 2 copy-number-changes every million years. Our results have shown that the observed cross-species variation among great-apes is significantly lower than expected when we extrapolate the mutation rate from within species variation (Wilcoxon rank-sum test, P-value < 0.01, Figure S7).

These simulations are simplistic as they do not take into account the effective population size of each species. Thus, we simulated genetic variation under a Wright-Fisher model of neutral evolution, taking into account both speciation and the process of coalescence. We used a combination of ms-based tools46 and the newly developed algorithm CoMuS (see methods for details) to obtain polymorphic tables for copy number variation data. We assumed a conservative range of theta (θ) values from 0.2 to 5.5 (i.e., corresponding to a range of mutation rates from 10−6–10−4 mutations per locus per generation, assuming Ne = 10,000) (Table S4). To take into account potential negative fitness effects due to extremely high PTS copy numbers, we incorporated a strong selection coefficient for copy numbers higher than 12. We chose this upper limit as it is the highest copy number we observed among primates. It is important to note that the inclusion or exclusion of this upper limit in our simulations does not change the trends we observed in this analysis. Using this approach, we generated the null distributions of copy number variation within and across species under neutrality. We then compared the neutral distribution of copy-number variation to the observed copy-number variation within and across-species variation for species where we have genotyped more than 3 individuals (i.e., 6 haplotypes) (Table S3). We found that the observed copy number variations within species (except for baboons) and among great apes are lower than the simulated variations (Fig. 4C). However between great apes and old world monkeys (vervets and baboons), we observed more-than-expected copy number variation as compared to simulated data. The increase in variation within baboons in Fig. 4C is due to the fact that all three individuals that we genotyped were heterozygous. It is plausible that further sampling would help to resolve this issue more conclusively. Overall, our results show that expansions or reductions of the copy number of MUC7 PTS-repeats occur mostly between different primate species. In contrast, within-species variation remains relatively stable.

Different adaptive pressures have likely shaped the extant genetic variation of MUC7 functional domains

To understand the adaptive forces shaping the non-repeated regions of the MUC7, we tested the presence of positive selection using the PAML package47 for each of the well-supported branches in the phylogenetic tree using MUC7 coding sequences outside of PTS-repeats across different mammalian clades (Figure S8). Our results suggest the action of positive selection on the branch leading to the primate ancestor for the MUC7 gene (PAML-codeml, P-value = 3.771 × 10−5, see Table S5 for the detailed results of the PAML analysis). The signal peptide of MUC7, which is important for transport of the protein outside of the cell14, remains highly conserved across mammals (Fig. 5), indicating negative selection. It is important to note that signal peptide is post-translationally cleaved from the mature MUC7 protein. In contrast, the histatin-like sequence of the gene, which is involved in antifungal activity48, shows a significant change in amino-acid sequences in primates as compared to non-primate mammals (Fig. 5). Indeed, when we individually tested these regions with PAML for positive selection, the histatin-like sequence, but not the sequence that codes for the signal peptide of the gene, showed evidence for positive selection (P-value = 0.002) in the primate lineage (Table S5). Nevertheless, the non-repeated N- and O-glycosylated sections of the gene, which include many alignment-gaps, as well as a high level of sequence diversity, retain the general “bottle brush-like” structure14. Briefly, the bottle brush-like structure refers to a high proportion of T and S amino-acids, which are potential targets for O-glycosylation, punctuated with P amino acids, which are important for maintaining the rigidity of the protein. This layout would then lead to a rigid linear structure with multiple O-glycans attached to this structure. We also observed relatively high level of change in the 3′-end of the gene, which encodes for a leucine-zipper motif in humans14. The domain may contribute to the overall stability of the highly glycosylated structure of MUC7 protein49 while the PTS-repeats determine the overall O-glycosylation of the protein. The leucine-rich domains of the protein seem to have evolved in the Simian lineage after the divergence of tarsiers (Fig. 5), and as such may have attained a relatively recent functional relevance in primates.

Figure 5: The adaptive evolution of MUC7 in mammals.
Figure 5

The upper-left panel shows the normalized ratio of nonsynonymous to synonymous variation for each functional segment of MUC7. The upper-right panel shows the overall nucleotide-level conservation for the same functional segments. The protein alignments of the signal peptide and the histatin-like domain that underlie these graphs are shown at the bottom. We did not include the PTS-repeat region in this analysis because copy number changes and sequence similarity introduce major alignment problems. P-values were calculated using a one-tailed Wilcoxon rank-sum test.

Discussion

We documented extensive genetic variation in MUC7 that has been shaped by multiple, species-specific adaptive constraints. Concordant with its role as an innate immunity component, MUC7 was reported to interact with a multitude of oral microorganisms and extra-oral pathogens50,51,52. Therefore, the increased MUC7 genetic diversity in primates could be a consequence of diverse pathogenic pressures, as suggested by the Red Queen hypothesis53.

Our results draw an initial picture of the evolution of MUC7 at three levels corresponding to three functional roles of the protein. At the first level, the signal peptide has evolved under negative selection, maintaining the extracellular activity of the protein. At the second level, the histatin-like domain has evolved under directional selection in the primate lineage, possibly to counteract a particular fungal pressure. At the third level, the copy number of PTS-repeats has evolved under lineage-specific adaptive constraints, likely determining the O-glycosylation repertoire of the protein, which is important for rheological and microbial binding properties of MUC752,54.

MUC7 provides a glimpse into adaptation through recurrent evolution of subexonic repeats. Copy numbers of most subexonic repeats normally remain highly conserved among mammals4, implying the strong influence of negative selection. The few subexonic repeats that remain copy number variable within species provide a distinct adaptive fodder for rapid evolution as hypothesized elsewhere55,56. First, the copy number of these exonic repeats changes rapidly57. Second, copy number variations of these sequences affect larger portions of the proteins than single nucleotide variations. Therefore, directional positive selection can lead to rapid increase or contraction of subexonic repeats in a species-specific manner. Examples of such rapid evolution of copy number variation of subexonic repeats in humans were demonstrated for the dopamine receptor gene58, as well as subexonic DUF1220 domain in multiple genes59. For MUC7, it is likely that the number of subexonic PTS-repeats, each carrying potential O-glycosylation sites, adaptively fine-tune the amount of O-glycosylation of the entire mucin protein in individual primate lineages. Indeed, similar mechanisms may play important roles in the evolution of other mucins. They may also affect the copy number variable cysteine-rich domains (CYS), which are functionally important in some mucin genes but apparently absent in MUC731. Overall, we anticipate that rapidly and recurrently evolving subexonic repeats similar to the ones of MUC7 will be discovered in other primate genes as targets of species-specific adaptation.

Methods

DNA Samples from non-human primate samples were purchased from Coriell Institute. The non-human primate samples that were genotyped can be found in Table S3. We used PCR to amplify the region that contains the entire PTS-repeat region of MUC7 in both human and other primates. The details for both experimental, bioinformatic and population genetic analysis can be found in below. We provide the R codes and sequence alignments that are used in this study in our website (gokcumenlab.org).

PCR Genotyping

PCR were performed in a total volume of 25 μl per reaction with 2.5 μl of 10 × standard Taq Reaction Buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2), 0.5 μl of dNTP (10 mM), 1 μl of MgCl2 (25 mM), 1 μl of each primer, 0.625 units of Taq DNA Polymerase (BioLabs Inc.), and 20–40 ng DNA template. The primers are listed in Table S2.

Bioinformatic and evolutionary analysis

To determine the presence of subexonic repeats across mammalian MUC7 orthologs, we used (i) BLAST (E-value < 0.01) to identify sequences that are similar to MUC7 human PTS-repeats and Simple Tandem Repeat track30, as well as through manual inspection. The genomic location information of MUC7 is on chr4: 71337834-71348714 (GRCh37/hg19) as defined by RefSeq gene track (NM_152291). Coding sequences of MUC7 in other mammals were extracted from GenBank60. The alignments were conducted both by Clustal Omega61 and Muscle62, followed by manual curation to reduce alignment errors. The conservation of MUC7 amino acid sequence was calculated by using Jensen-Shannon Divergence-based method63. The T and S amino acid content for MUC7 among different species was calculated by MEGA 6.0 after alignment64. A maximum-likelihood tree was built with MUC7 sequences across mammals using MEGA 6.064 under JTT with freq. (+F) model, and gamma distributed with invariant sites (G+I) with 500 bootstrap replicates (Figure S7).

Copy number simulations

We simulated the copy number changes for Great apes (Human, Chimpanzee, Gorilla and Orangutan) under different copy number gain-loss rate (0.5/1.0/1.5/2.0 copies per million year). Based on the species phylogeny of these four great ape species, we assumed that the original copy number state for the common ancestor of Great apes was 5 copies 11 million years ago. Then every 1 million year, there is a random gain or loss of 0.5/1.0/1.5/2.0 copies for Orangutan and the common ancestor of Human, Chimpanzee and Gorilla separately. At 8 million years ago, the common ancestor of Human and Chimpanzee separated from Gorilla and they started the copy number gain and loss simulation separately. The same simulation continues to 5 million years ago that Human and Chimpanzee separated from each other, and start their copy number gain/loss process independently until present. We simulated this process 1,000 times for 4 different copy-number-change rates (0.5/1.0/1.5/2.0 copies per million year), and for each simulation, calculated the variation of final state of simulated copy numbers for Human, Chimpanzee, Gorilla and Orangutan. The observed copy number state in present is Human 5 or 6 copies, Chimpanzee 5 copies, Gorilla 4 or 5 copies, and Orangutan 6 or 7 copies. The variations of observed copy number state in present are significantly less than variations simulated under neutrality (wilcoxon rank-sum test, P-value < 0.01).

We used CoMus (Coalescent of Multiple Species, http://pop-gen.eu/wordpress/software/comus-coalescent-of-multiple-species) to obtain within- and between-species polymorphisms. Assuming that k mutational events (gain or loss of copies) occur per million years, θ value is calculated by 4 × 25 × 104 × k/106 = k (effective population size for humans 104, and the generation time is set to 25 years). In our simulations, k varied between 0.2 to 5.5 mutational events per million years. Furthermore, we obtained a phylogenetic tree for Human, Chimpanzee, and Gorilla from the UCSC website (http://www.ucsc.edu/) and used it as a reference tree for the simulations. We assumed that the effective population size (Ne) is constant for each primate species and that Ne for Chimpanzees is twice the human Ne. Given a reference tree, and population parameters, CoMuS generates polymorphic samples similar to Hudson’s ms46. Then, we used microsat.c code provided in the ms software package to convert polymorphic samples to copy-number polymorphisms. Simulated datasets were used to obtain the null distribution of copy-number variation. Then, P-values were calculated by comparing the observed copy-number variability to the simulation-generated null distribution. This calculation was performed with the supposition that a copy number above 12 PTS-repeats is maladaptive, given that this is the highest copy number observed among primates. It is important to note that our results regarding the adaptive constraints on the PTS-repeats are partially dependent on the mutation rate of subexonic repeats, which have been shown to vary widely4,65.

Analysis for gene with salivary-specific expression

The dataset from gene-expression database, GTEx38 was used for the expression analysis (GTEx Analysis V4/RNA seq/Gene RPKM). We compared the expression in minor salivary gland to the expression in all other glands (n = 52) for all the genes in the human genome. On chromosome 4, Wilcoxon rank-sum test was applied to find the genes with significantly higher than expected expression (upper 1% of all the P-values) in salivary-gland than in other glands (n = 36 on chromosome 4), and we named them genes with salivary-specific expression. Then, we used a dynamic window approach to assess the density of salivary-specific expressed genes in every genomic region. Each window was selected to include exactly 15 genes. MUC7 locates in the cluster with the largest number of genes with salivary-specific expression in chromosome 4 (Fig. 2A,B).

PAML

To investigate deviations from neutrality in mammalian lineages, we conducted PAML47 using codeml program under branch site model A (allowing ω values to vary both among site and across branches), and compared with null model with fix ω = 1. Each branch in the phylogeny was tested separately. The difference of the -lnL values were compared by Chi-square test, and alpha-value = 0.05 (if P-value < 0.05 we reject the null hypothesis). The data can be found in Table S5.

Additional Information

How to cite this article: Xu, D. et al. Recent evolution of the salivary mucin MUC7. Sci. Rep. 6, 31791; doi: 10.1038/srep31791 (2016).

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Acknowledgements

The authors thank Andrew Clark, Derek Taylor, Paul Cullen and James Berry for critical readings of previous versions of this manuscript. This study is primarily funded by OG’s start-up funds, as well as IMPACT grant from the University at Buffalo Research Foundation. P.P. was funded by grants FP7 REGPOT-InnovCrete (Νο. 316223) and FP7-PEOPLE-2013-IEF EVOGREN (625057). S.R. is funded by NIH grants R01DE019807 and R21DE025826.

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Affiliations

  1. Department of Biological Sciences, State University of New York at Buffalo, New York 14260, USA

    • Duo Xu
    • , Emilie Redwood
    • , Sara Fox
    •  & Omer Gokcumen
  2. Institute of Computer Science (ICS), Foundation of Research and Technology–Hellas, Heraklion, Crete, Greece

    • Pavlos Pavlidis
  3. Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo, New York 14214, USA

    • Supaporn Thamadilok
    •  & Stefan Ruhl
  4. Department of Genetics, Cell Biology, and Development, University of Minnesota, Twin Cities, Minnesota 55455, USA

    • Ran Blekhman

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Contributions

O.G. and S.R. designed the experiments, oversaw the project and wrote the main manuscript text. D.X. conducted majority of the main analyses and prepared figures. P.P. conducted simulations for copy number variation evolution. R.B. helped analyze the evolutionary genetics analyses. D.X., S.T., E.R. and S.F. conducted P.C.R. experiments and conducted phylogenetic analyses of the data. All authors reviewed the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Stefan Ruhl or Omer Gokcumen.

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