Molecular Evolution and Protein Structure Variation of Dkk Family

Dkks have inhibitory effects on the Wnt signaling pathway, which is involved in the development of skin and its appendages and the regulation of hair growth. The nucleotide sequences were compared and analyzed to further investigate the relationship between the structure and function of the Dkk gene family and vertebrate epidermal hair. The analysis of the molecular evolution of the Dkk family revealed that the evolution rate of the genes changed significantly after speciation, with the Aves and Reptilia branches showing accelerated evolution. Additionally, positive selection was observed at specific sites. The tertiary structure of the protein was also predicted. The analysis of the functional divergence of the Dkk family revealed that the functional divergence coefficient of each gene was greater than 0, with most of the functional divergence sites were located in the Cys-2 domain and a few in the Cys-1 domain. This suggests that the amino acid and functional divergence sites may play a role in regulating the binding of the Dkk family to LRP5/6, and thus affect the inhibition of Wnt signaling, leading to different functions of Dkk1, Dkk2, and Dkk4 in the development of skin hair follicles. In addition, the Dkk families of Aves and Reptilia may have undergone adaptive evolution and functional divergence.


Introduction
The Dkk gene encodes a protein that acts as an inducer in the head of Xenopus laevis, and Dkk was found to be a potent antagonist of Wnt signaling [1].After that, Dkk genes were found in vertebrates and invertebrates one after another [2][3][4][5].The Dkk gene family encodes secreted proteins, such as Dkk1-4 and soggy (Sgy), which are predominantly found in vertebrates.Four Dkk proteins (Dkk1-4) exist in humans, and all contain two cysteine-rich domains (CRDs), designated CRD1 and CRD2, each of which contains five disulfide bonds [6].Members of the Dkk protein family can bind to the co-receptor of Wnt, namely low-density lipoprotein receptor-related protein (LRP), and regulate its activity, thus controlling the transduction of Wnt signals.Sgy is a novel secreted protein related to Dkk-3 but which lacks the cysteine-rich domains [2].The function of Sgy is different from that of the other Dkk family members.The Dkk family of proteins is known to play a role in regulating the activity of the Wnt signaling pathway, which is involved in the development and regeneration of hair follicles.By modulating the activity of the Wnt signaling pathway, the Dkk family can influence the development and periodic regeneration of hair.
Studies have found that the blocking effect of Dkk1 occurs at the beginning of epidermal morphological changes or cell differentiation caused by molecular signals [7].A study found that ectopic expression of Dkk1 in the epidermis leads to defects in tentacles, Genes 2023, 14, 1863 2 of 12 hair, teeth, and mammary glands [8].These studies suggest that Dkk1 negatively regulates hair follicle development and follicle number by antagonizing the canonical Wnt signaling pathway.Dkk2 inhibits the formation of plantar hair follicles and maintains the normal shape of plantar skin in embryonic development by blocking the Wnt/β-catenin signaling pathway [7].When Dkk2 is overexpressed, the activity of the dermal papilla is reduced; when Dkk2 is knocked out, pulp formation is reduced, both of which lead to delayed feather regeneration [9].These indicate that knockout and overexpression of Dkk2 could not maintain the periodic regeneration of hair follicles.Dkk3 has not been reported to be related to hair follicles.The expression of Dkk4 was higher in primary hair follicles, but significantly decreased in secondary hair follicles and growing hair follicles [10].Cui prepared skin-specific Dkk4 transgenic mice to study the role of Dkk4 in hair follicle development.In another experiment, the introduction of Dkk4 into cats and wild-type mice had no effect on primary hair, but the induction of secondary hair and hair follicles was completely prevented [11].
Current research has revealed that the Dkk gene family plays a critical role in skin morphology, hair follicle development and growth cycle, and embryonic development of tissue and organs.Vertebrate skin appendages include scales, feathers, and hair; although they look different, they are regulated by the Wnt signaling pathway.This study focused on the functional divergence and molecular evolution of the Dkk gene family in the context of skin appendage evolution.In order to gain a better understanding of the genetic variation and evolutionary relationship of the Dkk gene family in vertebrates, we retrieved the coding region sequences of Dkk gene family members from the Genebank database on the NCBI website for a selection of representative species.Because the structure and function of sgy is different from that of Dkk1-4, we only analyze Dkk1-4.The gene sequences of Dkk1-4 were then analyzed using molecular evolution techniques to gain insights into their genetic variation and evolutionary relationship.Additionally, the protein structures of these genes were also analyzed to gain further insights into their function and role in vertebrate evolution.

Sequence Alignment and Phylogenetic Analyses
To study the phylogenetic relationship between the different genes, a phylogenetic tree was constructed using the Maximum Likelihood method.From the topology of the tree, it can be seen that Dkk1, Dkk2, and Dkk4 all originated from Dkk3 (Figure 1).Multiple sequence alignments were performed for the Dkk1-4 genes.There are obvious differences in the gene sequence of Dkk1 in vertebrates; Aves lack 10 amino acids in the middle region (Figure S1A).This study also found that the protein structure in Aves is different from that of humans and mice (Figure 2).From the tertiary structure of proteins, it can be observed that avian protein sequences are shorter and lack a significant amount of α-helices.In the multiple sequence alignment of Dkk2, there were differences between Aves and Reptilia: 14 amino acids were inserted in Aves and Reptilia (Figure S1B).In the multiple sequence alignment of Dkk3, there are differences among Aves, Reptilia, Anura, and Chiroptera; 11 amino acids were inserted in Aves and Reptilia (Figure S1C).The results of the multiple sequence alignment of Dkk4 showed no significant differences.

Variation in Molecular Evolution Rate of Dkk
To examine the changes in selection pressure on the four genes of the Dkk gene family during the evolution of vertebrates, we calculated the ω values using the model M0 in PAML (Phylogenetic Analysis by Maximum Likelihood): ω Dkk1 = 0.10793, ω Dkk2 = 0.06302, ω Dkk3 = 0.20621, and ω Dkk4 = 0.26750.The results show that the synonymous substitution rate is much higher than the non-synonymous substitution rate, indicating that the entire Dkk gene family has generally undergone purifying selection during the evolution of vertebrates, showing the functional conservation of this gene family.
Although the overall Dkk gene sequence was strongly purified and selected, it could not be ruled out that some amino acids at specific sites were under positive selection.Therefore, we used Site models (M3 vs. M0 and M2 vs. M1) to detect the selection pressure at all sites.The LRT difference between M3 and M0 of Dkk1 was significant (2∆l = 10858.42,df = 4, p < 0.001), indicating that discrete selection pressures were applied to different sites of Dkk1, but none of the sites were in a positive selection state.The LRT between M1 and M2 of Dkk1 does not support the hypothesis that model M2 is superior to model M1 (2∆l = 0, df = 2, p > 0.05), and there is no positive selection site.The results of each model are shown in Table S2.The results showed that all Dkk genes were under purifying selection, and that no positive sites were found.Although all four genes showed purifying selection, a free-ratio model was developed in order to better reflect the selection pressure of different species in evolution.The results showed that in Dkk1, the ω value of Reptilia (ω = 21.85) was significantly higher than that of other branches.In Dkk3, Aves and Reptilia have ω = 3.04.However, in Aves species, ω = 999 for the Dkk1 and Dkk3 genes.In Dkk2 and Dkk4, the ω-value of the branch of Pholidota and Carnivora was significantly higher than that of the other branches (ω > 1).A ω ratio significantly greater than one is a convincing indicator of positive selection.These results suggest that positive selection may have played a potential role in the early evolution of Reptilia, Aves, Pholidota, and Carnivora.
To further determine if any amino acid sites in the Dkk gene family's accelerated evolution branch are under selection pressure, we ran Model A (Model = 2, NSsites = 2) in the branch site model, which takes into account not only the ω value between sites but also the ω value between branches.The test results (Table 1) are all based on the probability calculated by the BEB method (*: p > 95%; **: p > 99%).

Dkks Functional Divergence Analysis Results
According to the previously constructed species evolution tree, the vertebrates were divided into three groups: A for Mammalia, B for Reptilia, and C for Aves (Tables 2 and 3).Most of these functional divergence sites are distributed in the Cys-2 domain, and very few are distributed in the Cys-1 domain.In the Dkk gene family, Dkk2, Dkk3, and Dkk4 had obvious type I functional divergence; the value of θ I was between 0.23 and 0.63, while the θ I coefficient of Dkk1 was relatively small.The θ II coefficients of Dkk1-4 are all relatively small (Tables 2 and 3).

Dkk Protein Analysis Results
To infer structure-function correlations, the sequences of the positive selection sites were detected using the PAML software and are based on the human amino acid sequence.This is because some amino acids will be deleted when the PAML software is running, so it is necessary to determine the position of the detected amino acid position in the complete amino acid sequence.The pictures of the involved sites are shown in Figure S3.We found that there was a change in the protein sequence of Homo sapiens (255S) and Anas platyrhynchos (223P) Dkk1, and the protein structure changed from a turn to a coil.This amino acid site is located in the Cys-2 domain.In Dkk2, the amino acid site (27V) of H. sapiens and the corresponding amino acid site of Manis pentadactyla (86M) changed, and the protein structure changed from a turn to a coil.This amino acid site is located in the Cys-1 domain.In Dkk3, the amino acid site (264R) of H. sapiens and the corresponding amino acid site of Zonotrichia albicollis (209L) changed, and the protein structure changed from a turn to a coil.In Dkk4, the amino acid site (132K) of H. sapiens and the corresponding amino acid site of Podarcis muralis (131Q) changed, and the protein structure changed from a coil to a turn.

Discussion
In this paper, we investigated the evolutionary relationship of Dkk proteins in vertebrates.We discuss the nature of Dkk's interactions with its partner Krm1 and the E3E4 region of LRP5/6, which have been widely established, and the functional divergence of Dkk proteins in vertebrate evolution, which has not been reported.We also explore the surprising lack of understanding of the Dkk family in terms of molecular evolution.Our findings provide insight into the evolution of Dkk proteins and their role in Wnt signaling.
In the multiple sequence alignment, Dkk1, Dkk2, and Dkk3 showed obvious differences.The inserted amino acids have an irregular curl in the protein structure.The insertion of these amino acids may cause the Dkk genes to differ in skin phenotype between different species.Through the construction of a phylogenetic tree, the phylogenetic relationship between species or genes can be displayed clearly.Dkk1, Dkk2, and Dkk4 all originated from Dkk3.A study confirmed that vertebrate Dkk-1, 2, and 4 may have originated from a common ancestor gene of Dkk3 [12].But, in another study, Dkk3 appears to be a divergent member of the Dkk family [13].The origin of the Dkk family is currently debated, but our results confirm that they originate from Dkk3.Due to structural differences, Dkk3 proteins exhibit biological characteristics that are different from those of the other family members.Most of the reports on Dkk3 gene are closely related to the occurrence, development, metastasis, and prognosis of common tumors [14].Dkk1, Dkk2, and Dkk4 have all been reported to be involved in hair follicle development.The Dkk family may play an important role in hair follicle variation in vertebrates.To test this hypothesis, we used CodeML estimates of synonymous and non-synonymous substitutions.
The results of the M0 model indicate that the Dkk gene family is under purifying selection in most vertebrates.The M0 model indicates that this gene family has important functions.In addition, the free-ratio model evolution studies have shown that, in vertebrates, the selection pressure of the Dkk family changes.The adaptive evolution of the four genes occurred primarily in single branches of the phylogenetic tree, including Reptilia, Aves, Cetaceans, Lepidoptera, and Carnivora.For Reptilia and Aves Dkk1 and Dkk3 genes, ω > 1.An ω ratio significantly higher than 1 is convincing evidence of diversification [15].Aves Dkk1 and Dkk3 showed the strongest positive selection signal (ω = 999) based on the branch mode.Reptilian scales and avian feathers are considered homologous structures [16].The complex topology of bird feathers may be responsible for the accelerated evolution of birds during feather development.The topology of bird feathers is more complex than that of reptile scales [17].Feathers consist of many tiny structures that form complex interactions and liaisons between each other.This complex structure allows birds to be more adaptable in terms of flight and protecting themselves.However, in the evolution of vertebrates, the hair phenotypes of Reptilia and Aves are unique.Given that diversity in hair development can occur through multiple pathways, this lack of a parallel signature is perhaps not surprising [18].The results of the Branch site model of the Dkk gene family showed that there were adaptive evolution and purifying selection sites in Reptilia in Dkk1, Dkk3, and Dkk4.There are also purifying selection sites in birds in Dkk1 and Dkk3.Moreover, the amino acid sites we found almost all existed in the CRD.It was found that the CRD domain is both necessary and sufficient for the binding to Wnt [19,20].However, the Wnt signaling pathway plays an important role in many genes or signaling pathways that regulate hair follicle growth and development [21,22].
To further determine whether the Dkk gene family had functional divergence among species, family members were tested for type I and type II functional divergence.The degree of functional divergence of type I is greater than that of type II.This indicates that the functional constraints between replicative genes have changed [23].Our results also proved that the Dkk gene family underwent accelerated evolution during species evolution, and some residues may have undergone functional restriction changes after speciation.Mammals, Aves, and Reptilia have different hair phenotypes, which may cause the genes to have different functions.At the same time, one purifying selection site (281V) in the Dkk3 gene was also identified as functional divergence site by the pressure selection analysis.
Most of the purifying selection sites and functional divergence sites screened in this study are in the middle and downstream regions, and some of them are in the Cys-2 domain.Dkk1, Dkk2, and Dkk4 have been identified as effective inhibitors of Wnt signaling and bind to the Wnt coreceptor LRP5/6 [24][25][26].However, some studies have found that CRD2 is essential for suppressing Wnt signals [27].The binding site of Dkk1 CRD2 to LRP5/6 has been reported [28,29].The binding sites of Dkk1 and Kremen1 have also been reported [30,31].Neutral sites were found near the binding sites.Mohammadpour performed in silico analyses and established that Dkk3, similar to other Dkk family members, can bind to the third PE pair of LRP5/6 through its CRD2 [32].In another in silico study, Fujii reported that the insertion of seven amino acids (L249-E255 in human Dkk3) and P258 reduced the binding affinity between DKK3 and LRP5/6 [33].Interestingly, the D250 residue in the Dkk3 protein sequence was mutated to N in Aves, Reptilia, and Amphibia (Figure 3).This mutation leads to a decrease in amino acid hydrophilicity.Comparing the tertiary structures of three species (H.sapiens, Zootoca vivipara, and Gallus gallus), the secondary structure of D250N changed from a turn to a random coil (Figure 4).When the hydrophilicity of an amino acid is weakened, it may have an effect on the structure and function of the protein [34].
reduced the binding affinity between DKK3 and LRP5/6 [33].Interestingly, the D250 residue in the Dkk3 protein sequence was mutated to N in Aves, Reptilia, and Amphibia (Figure 3).This mutation leads to a decrease in amino acid hydrophilicity.Comparing the tertiary structures of three species (H.sapiens, Zootoca vivipara, and Gallus gallus), the secondary structure of D250N changed from a turn to a random coil (Figure 4).When the hydrophilicity of an amino acid is weakened, it may have an effect on the structure and function of the protein [34].A study designed and improved several small peptides based on the LRP6-binding site of the CRD2 of Dkk3 [35].These peptides were highly capable of binding to LRP6 in silico, and may prevent the formation of an active Wnt-LRP6-Fz complex [35].In the experiment conducted by Poorebrahim, several small peptides did not alter 264R, an amino acid that forms a salt bridge with Asp811 of LRP6.In our experiment, we found a mutation in 264R in the Aves species, where Arg was replaced by Gln, resulting in decreased hydrophilicity (Figure 3).In the protein structure, two amino acid residues with opposite charges form an ion pair; when the distance between the charged groups of the two amino acid side chains in the ion pair (that is, any oxygen atom in the negatively charged residue carboxylate and the positively charged residue side or the distance between any nitrogen atom in the chain) is less than 4 Å, the ion pair is considered a salt bridge [36,37].Gln is a polar uncharged residue.The Arg264Gln substitution would abolish a salt bridge with Asp811 in LRP6.The selected amino acids found in CRD2 may affect the binding of the Dkk gene family to LRP5/6, thus affecting the inhibition mechanism of Wnt signals and making Dkk1, Dkk2, and Dkk4 show different functions in hair follicle development.Vertebrate habitats range from the deepest parts of the ocean to the highest peaks of mountain ranges, from the tropics to the Arctic.The environmental variations trigger divergent natural selection, leading to the emergence of niche specialists [27].The living environment of Aves and Reptilia may also cause these genes to differentiate in function during the evolutionary process.
enes 2023, 14, x FOR PEER REVIEW 8  A study designed and improved several small peptides based on the LRP6-bi site of the CRD2 of Dkk3 [35].These peptides were highly capable of binding to LR silico, and may prevent the formation of an active Wnt-LRP6-Fz complex [35].In th periment conducted by Poorebrahim, several small peptides did not alter 264R, an a acid that forms a salt bridge with Asp811 of LRP6.In our experiment, we found a mu in 264R in the Aves species, where Arg was replaced by Gln, resulting in decrease drophilicity (Figure 3).In the protein structure, two amino acid residues with op charges form an ion pair; when the distance between the charged groups of the two a acid side chains in the ion pair (that is, any oxygen atom in the negatively charged re carboxylate and the positively charged residue side or the distance between any nit atom in the chain) is less than 4 Å, the ion pair is considered a salt bridge [36,37].G polar uncharged residue.The Arg264Gln substitution would abolish a salt bridge Asp811 in LRP6.The selected amino acids found in CRD2 may affect the binding Dkk gene family to LRP5/6, thus affecting the inhibition mechanism of Wnt signal making Dkk1, Dkk2, and Dkk4 show different functions in hair follicle development tebrate habitats range from the deepest parts of the ocean to the highest peaks of mou ranges, from the tropics to the Arctic.The environmental variations trigger divergen ural selection, leading to the emergence of niche specialists [27].The living environ of Aves and Reptilia may also cause these genes to differentiate in function durin evolutionary process.

Conclusions
Dkk1-4 all underwent accelerated evolution and purifying sites were detected

Conclusions
Dkk1-4 all underwent accelerated evolution and purifying sites were detected.The changes in the Dkk gene family in vertebrates under selection pressure and functional divergence were tested.However, the evolution rates of purifying selection sites and functional divergence sites are different.These amino acid sites will affect the tertiary structure of proteins and make genes differentiate functionally.The current study shows that the Dkk gene family underwent changes in selection patterns during vertebrate evolution and may have acquired additional functional constraints in different branches.

Sequence Acquisition
Sequence data of the CDS of the Dkk1-4 genes of different species were retrieved from the GenBank database.For the Dkk1 gene, a total of 45 species were selected, 47 species were selected for the Dkk2 gene, 47 species for the Dkk3 gene, and 34 species for the Dkk4 gene (Table S1).These data were then used to analyze the genetic variation and evolutionary relationship of the Dkk gene family in vertebrates.Hydra magnipapillata is an invertebrate.We chose Hydra as an outgroup because we wanted to study the origin of the Dkk gene family.The Dkk gene family has been found in vertebrates and some invertebrate phyla but Dkk4 appears to only be present in mammals and Reptilia.

Nucleotide Sequence Analysis
Using MEGA-X [38], the Maximum Likelihood tree of the amino acid sequences of the Dkk gene family of vertebrates was constructed, and the confidence value of each branch was calculated with 500 repetitions of the Bootstrap test.Additionally, the nucleotide sequences of the Dkk1-4 genes of the above species were aligned using the ClustaW

Genes 2023 , 12 Figure 1 .
Figure 1.Phylogenetic tree of Dkk gene family.The numbers on the evolutionary tree are bootstrap values.Figure 1. Phylogenetic tree of Dkk gene family.The numbers on the evolutionary tree are bootstrap values.

Figure 1 .
Figure 1.Phylogenetic tree of Dkk gene family.The numbers on the evolutionary tree are bootstrap values.Figure 1. Phylogenetic tree of Dkk gene family.The numbers on the evolutionary tree are bootstrap values.

Figure 1 .
Figure 1.Phylogenetic tree of Dkk gene family.The numbers on the evolutionary tree are bootstrap values.

Figure 2 .
Figure 2. Protein structure of human, mouse, pigeon, and burrowing owl Dkk1.Deep red represents α-helix, yellow represents β-fold, light blue represents coil, and white represents other residues.

Figure 3 .
Figure 3. Dkk multiple sequence alignment.Highly similar residues are colored in red and framed in blue.The green number indicates the disulfide bond.The yellow box at the bottom of the sequence represents the residue involved in the interaction between Dkk1 and LRP6.The brown triangle is the residue involved in the interaction between Dkk1 and Kremen proteins.Seven amino acids (L249-E255 in human Dkk3, LDLITWE) and P258 are represented in a purple box.In the purple box, the black letter is the site of a Dkk3 mutation (D250N).The green box is the Arg264Gln of Dkk3 gene.Abbreviations: H = H.sapiens, M = Mus musculus, G = G. gallus, A = Anas platyrhynchos, C = Columba livia, P = Pygoscelis adeliae, and Z = Z.vivipara.

Figure 3 .
Figure 3. Dkk multiple sequence alignment.Highly similar residues are colored in red and f in blue.The green number indicates the disulfide bond.The yellow box at the bottom of the seq represents the residue involved in the interaction between Dkk1 and LRP6.The brown tria the residue involved in the interaction between Dkk1 and Kremen proteins.Seven amino (L249-E255 in human Dkk3, LDLITWE) and P258 are represented in a purple box.In the purp the black letter is the site of a Dkk3 mutation (D250N).The green box is the Arg264Gln of Dkk3 Abbreviations: H = H.sapiens, M = Mus musculus, G = G. gallus, A = Anas platyrhynchos, C = C livia, P = Pygoscelis adeliae, and Z = Z.vivipara.

Figure 4 .
Figure 4. Overlay structure of Dkk3 proteins from three species.Species: H. sapiens (brown), Z vivipara (light blue), and G. gallus (pink).Red, green, and dark blue represent the relative po of the D250N mutation.The secondary structure of 250N in Z. vivipara is a random coil.

Figure 4 .
Figure 4. Overlay structure of Dkk3 proteins from three species.Species: H. sapiens (brown), Zootoca vivipara (light blue), and G. gallus (pink).Red, green, and dark blue represent the relative positions of the D250N mutation.The secondary structure of 250N in Z. vivipara is a random coil.

Table 1 .
Results of branch site model.

Table 2 .
Functional divergence of type I Dkk gene family.

Table 3 .
Functional divergence of type II Dkk gene family.