There have been no studies around the in-silico approach to papain function in the regulation of abnormal scar formation. Based on this study, papain has the strongest homology with Cathepsin K compared to Cathepsin B. Since Cathepsin K is a member of the papain superfamily and has greater capability to degrade collagen type I than Cathepsin B, we suggest that papain is a potential candidate for the treatment modality of abnormal scars.

An abnormal scar is formed due to improper management of a wound and impacts patients both in terms of aesthetics as well as functionality^1,2. Various factors cause the formation of an abnormal scar, including genetics of skin color (mainly in dark-skin races), trauma, excessive voltage strength which causes cell communication disruption, and imbalance between collagen synthesis and degradation^3,4. An abnormal scar is characterized by prominent growth of the scar tissue, redness due to neovascularization, and itching that is sometimes accompanied by pain⁵. Abnormal scars are divided into two types: the keloid scar and the hypertrophic scar. The keloid scar (from “chele” and “oid”) is a scar tissue that protrudes and spreads into normal tissue outside the edges of the wound with specific predilection sites. Meanwhile, the hypertrophic scar does not spread outside the wound^6-9.

The abnormal scar can occur in men and women, most often between 11 and 30 years of age¹⁰. Epidemiological studies conducted (by Zhu et al.)¹¹ indicated that a total of 100 million patients in developing countries had an abnormal scar derived from elective surgeries (approximately 55million) and surgeries (approximately 25million). The prevalence of abnormal scars (hypertrophic and keloid) in Indonesia remains unknown; however, those with darker skin, such as Indians and Malays, tend to more vulnerable to developing an abnormal scar¹².

The therapeutic modalities for abnormal scars, such as excision surgery, silicon-based therapy or corticosteroid injection, do not provide optimal improvement owing to their side effects. Therefore, alternative therapy with minimal side effects is recommended over therapeutic modalities for the treatment of such scars. Papain has proteolytic anti-microbial properties that can dissolve dead cells and shorten wound healing time, reducing damage to healthy tissue surrounding the abnormal scar^13,14. Wihastyoko and Hanafi¹⁵ used one-way ANOVA (p <0.05) to prove papain’s potential to degrade excess collagen in abnormal scars in vitro and in vivo. Papain uses proteinase in catalytic activity, and thus, the active sides of papain are able to hydrolyze peptides. Moreover, the widespread availability of papaya makes it plausible to use papain as a new therapeutic modality¹⁶.

The mechanism of how papain degrades collagen, directly or indirectly, remains unknown. Investigating other enzymes with comparable biological activity and sequence similarity, such as collageneolytic, may provide insight into how papain works. Collageneolytic is an enzyme that has the ability to catabolize excessive collagen at affected site¹⁷.

Papain enzyme is a group of cysteine proteases that found in the Carica papaya plant. Cysteine proteases are part of a protease group that catalyze protein degradation through the hydrolysis of the peptide bond. This protease is categorized as zymogen or proenzyme; it is a group of enzymes synthesized as inactive precursors, with the ability to become active through changes in their biochemical environment. Cathepsin is another family of cysteine protease that is considered to be papain-like enzyme and is abundantly found in mammals^18,19. Human genomes encode 11 kinds of Cathepsins, namely Cathepsins B, C, F, H, K, L, O, S, V, and X²⁰.

Cathepsin B is an enzyme that belongs to the lysosomal cysteine proteases family. It possesses GNFD motifs, lacks ERFNIN motif, but has occluding loop feature. The occluding loop results in carboxypeptidase activity. Cathepsin B has a pro-domain with 62 residues and a mature domain with 260 residues. It is auto-activated using exopeptidase cleavage property¹⁹. In humans, Cathepsin B functions by removing amyloid plaques in people with Alzheimer’s disease and degrading extracellular matrix through matrix metalloproteinase (MMP)²¹. Cathepsin B is also able to degrade collagen at both acidic and neutral pH by regulating fibroblast through TLR2/NF-ĸB activation^22,23, indicating that Cathepsin B has a role in wound healing and tissue remodeling²⁴. However, the role of Cathepsin B in improving abnormal scar remains unknown.

Cathepsin K is a lysosomal cysteine protease involved in bone remodeling and resorption in humans. Unlike Cathepsin B, it has ERFNIN motif but no occluding loop feature. The pro-domain of Cathepsin K has 99 residues with 215 residues of mature domain. It is also auto-activated by exopeptidase properties, such as Cathepsin B, to perform catalysis at pH 4.0 or lower^19,25. Cathepsin K is dominantly produced in osteoblast and has the ability to degrade collagen I and II. It is also a major collagenolytic protease in bones and also effectively degrades other extracellular matrix components^25,26. Research around Cathepsin K has focused on bone development, but studies are lacking around its impact on wound healing and abnormal scar healing.

The ability of in silico analysis to manipulate biomolecular products can strengthen the results of in vivo and in vitro studies and be used as supporting data in the innovation of therapy modalities. Till date, there have been no studies around the in-silico approach to papain function in the regulation of abnormal scar formation. Thus, this study aims to identify the potential of papain as a candidate for the treatment modality of abnormal scar through in silico studies by comparing the biological activity and sequence similarity to Cathepsin B and Cathepsin K.

METHODS:

The direct or indirect process of collagen degradation through papain was investigated using several tools. To determine the potential of the direct mechanism, the cleavage site of papain was examined using MEROPS database by accessing http://merops.sanger.ac.uk/²⁷ with ID C01.001. The cleavage pattern was obtained in the option cleavage site specificity in the summary menus. The collagen sequence was obtained from the Uniprot database (www.uniprot.org) using the accession code P02542. Additionally, the collagen sequence in FASTA format was compared with papain cleavage site in MEROPS database to determine the potential of direct mechanism in degrading collagen. The potential of indirect mechanism on the collagen degradation process with papain was determined using String DB (string-db.org) and visualized using Cytoscape 3.8.0 tools.

Since Cathepsin B and Cathepsin K are human papain-like enzymes with comparable biological activity as papain in degrading collagen in extracellular matrix^28,29, we compared the ratio of similarity and identity of the human enzymes with papain. The sequences of Cathepsin B and Cathepsin K were retrieved from Uniprot using IDs P07858 and P43235. The percentage of similarity and identity values were obtained from sequence and structure analysis using Bioedit 7.2.

RESULTS:

The results of the active site analysis on the papain enzyme obtained from MEROPS peptidase database (Figure 1A) showed that papain recognizes QQ-D (Gln-Gln Asp) motif as the cleavage target, while collagen only has a QQ motif within its sequence (Figure 1B). The results of MEROPS cleavage site analysis have shown that the direct interaction approach between papain and collagen does not adequately explain the mechanism behind the degradation of collagen due to differences in the degradation pattern of the papain enzyme.

Fig 1: (A) The pattern of papain enzyme degradation with QQ-D (Gln-Gln_Asp) motifs; (B) The pattern of Collagen Degradation without QQ motifs.

Analysis using Bioedit found that papain had similar effects as Cathepsin B and Cathepsin K in degrading collagen. The homology result showed that Cathepsin K has a closer relationship with papain, as indicated by its higher identity and similarity results compared to Cathepsin B (Table 1, Figure 2 and Figure 3).

Table 1. Biodit analysis

Protein	Identity	Similarity
Cathepsin K >< Papain	36%	53%
Cathepsin B >< Papain	25%	38%

Fig 2: Homology of Cathepsin K protein and papain enzyme (A) Cathepsin K, (B) Papain, (C) Alignment visualization of Cathepsin K and papain

Fig 3: Homology of Cathepsin B protein and papain enzyme (A) Cathepsin B, (B) Papain, (C) Alignment visualization of Cathepsin B and papain

Thus, papain is more likely to have a similar mechanism to Cathepsin K in regulating collagen synthesis and degradation, thus inhibiting abnormal scarring. The Cytoscape result showed that papain interacts indirectly with COL1A1, as depicted by the protease activities of Cathepsin B and Cathepsin K (Figure 4).

DISCUSSION:

A motif is a sequence that indicates the presence of certain biological characteristics³⁰. This study suggested that papain might have an indirect interaction with collagen. We analyzed the suggested indirect interaction of papain with collagen by identifying the homologous proteins of papain in humans and investigating papain interactions with collagen in the DB String and cytoscape visualization.

A logo represents each column of the alignment through a stack of letters, with the height of each letter proportional to the observed frequency of the corresponding amino acid, and the overall height of each stack proportional to the sequence conservation, measured in bits, at that position (Figure 1A)³¹. These motifs indicate the referred amino acid residues in each cleavage site. Q denotes Gln amino acid and D denotes Asp amino acid.

COL1A1 is a gene that encodes and is a major component in type I collagen^32,33. This result indicates that Cathepsin K has the potential to degrade collagen type I trough interaction with the COL1A1 gene. Cathepsin B also has the ability to interact with COL1A1 via ubiquitin (UBC) proteasome.

Papain’s characteristic as a proteolytic agent could degrade the extracellular matrix through indirect contact with collagen on its active side or through the ubiquitin–proteasome complex, specifically the 26S proteasome, using Ubiquitin C (UBC), which works intracellularly, as an intermediary. Protease enzymes, including papain, degrade the target protein through Ubiquitin–Proteasome Pathway (UPP) on the active site of the catalyst^34-36. According to this research, Cathepsin B has the ability to degrade collagen type I by interacting with COL1A1 through the UBC mechanism.

Cathepsin K is an enzyme located in the lysosome and plasma membrane (apical cell membrane, peripheral membrane, and extracellular side)³⁷. Research by Boraschi-Diaz et al.³⁸ demonstrated that Cathepsin K can hydrolyze collagen type I. This enzyme can cleave the collagen in the triple helix position of multiple locations and can be determined in the N-terminal position^39,40.

Papain is an enzyme obtained from the sap of the papaya plant (Carica papaya L.). The enzyme is also known as papayatin or vegetable pepsin because it has a similar mechanism to pepsin and trypsin in its ability to digest proteins. Papain contains 212 amino acids in a cross-linked polypeptide chain with three disulfide bridges and six sulfhydryl groups, with two sites of active sulfhydryl groups. Papain is a proteolytic enzyme that catalyzes peptide bonds into small compounds such as dipeptides and amino acids¹⁶. Papain’s catalytic activity is carried out through hydrolysis on its active side, resulting in a proteinase that is capable of hydrolyzing peptides. Papain hydrolysis in most protein substrates is more extensive than other proteases such as trypsin and pepsin ⁴¹. Papain activity is influenced by many factors, such as pH temperature, ion strength, pressure, and the active side containing the sulfhydryl group. According to Suhartono⁴², the optimum pH of papain is 4.5–7 and optimum temperature is 60–75^oC.

Papain has been used as a therapeutic modality for abnormal scars for a long time, albeit without clear data on its mechanism of action. Junior et al. ⁴³ and Ramundo and Gray⁴⁴ explained that papain could help degrade the necrotic tissue in the wound, resulting in shorter healing time without damaging the healthy/vital tissue around the lesion. Papain was believed to be a therapeutic modality for maintaining the regulation of the synthesis and degradation of collagen to prevent formation of abnormal scars.

Papain acts as a debridement agent involved in the breakdown of the polypeptide chain and/or hydrolysis of the collagen cross-linkage. This stabilizes the collagen fibrils, which was weaker and more vulnerable when exposed to papain gel⁴⁵. The papain enzyme had the ability to stimulate Matrix Metalloproteinase-2 (MMP-2) activity through zymography in human fibroblast cells^[46,47]. MMP-2 required the stimulation of fibroblasts and macrophages against plasminogen, which converts to plasmin through activator tissue-Plasminogen Activator (t-PA) and urokinase-Plasminogen Activator (u-PA) in order to degrade collage (7,48)⁴⁸. Chen et al.⁴⁹ explained the therapeutic effect of the papain enzyme in the form of Papain Elastic Liposome (PEL), which had the best results with the Encapsulation Efficiency (EE) formulation of (43.8±1.4%), particle size (100.9±2.2 nm), PDI (0.037±0.003), zeta potential (-26.3±1.3 mV), and Deformability Index (DI) (21.9±3.1). The most stable cumulative number and fluxes were in solution 381.9±32.4μg/cm2, 11.4±1.5μg/cm²/h, and drug deposition in the skin 19.1±3.2% after 24 hours.

CONCLUSION:

The in-silico test of papain in degrading collagen using MEROPS/Peptidase Database, Bioedit, String DB, and Cytoscape suggested that papain might have an indirect interaction with collagen type I (COL1A1). It also showed that papain has the strongest homology with Cathepsin K compared to Cathepsin B. Since Cathepsin K is a member of the papain superfamily and has greater capability to degrade collagen type I than Cathepsin B, we suggest that papain is a potential candidate for the treatment modality of abnormal scars.

The limitations of this research include the similarity between papain, Cathepsin K, Cathepsin B, and their interaction in collagen I degradation. Further research needs to be conducted since papain has a large Cathepsin family that could be applied as a new therapeutic modality to degrade other kinds of collagen, which play a role in the treatment of abnormal scarring.

AUTHOR CONTRIBUTION:

HYLW: Leading author, HYLW, SS: Manuscript writing; EW, K: Concept, critical revision; K, BP: critical revision,

CONFLICT OF INTEREST:

The authors state that there are no conflicts of interest regarding the publication of this article.

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Received on 13.10.2020 Modified on 09.11.2020

Research J. Pharm. and Tech. 2021; 14(9):4957-4962.

DOI: 10.52711/0974-360X.2021.00862