Collagen fibrils of sea cucumber (Apostichopus japonicus) are heterotypic

Journal Pre-proofs Short communication Collagen fibrils of sea cucumber (Apostichopus japonicus) are Mo Tian, Changhu Xue, Yaoguang Chang, Jingjing Shen, Yuying Zhang, Zhaojie Li, Yanchao Wang PII: DOI: Reference:

S0308-8146(20)30120-5 https://doi.org/10.1016/j.foodchem.2020.126272 FOCH 126272

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Food Chemistry

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12 June 2019 18 January 2020 19 January 2020

Please cite this article as: Tian, M., Xue, C., Chang, Y., Shen, J., Zhang, Y., Li, Z., Wang, Y., Collagen fibrils of sea cucumber (Apostichopus japonicus) are, Food Chemistry (2020), doi: https://doi.org/10.1016/j.foodchem. 2020.126272

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1

Collagen fibrils of sea cucumber (Apostichopus japonicus) are

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Mo Tiana, Changhu Xuea,b, Yaoguang Changa,b*, Jingjing Shena, Yuying

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Zhanga, Zhaojie Lia, Yanchao Wanga

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a.

6

China

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b.

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Science and Technology, Qingdao, 266237, China

College of Food Science and Engineering, Ocean University of China, Qingdao, 266003,

Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine

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Journal: Food Chemistry

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Submitted: June 2019

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*Corresponding

author. E-mail address: [email protected]; Tel.: + 86-532-82032597

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1

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Abstract

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Sea cucumbers attracted increasing interest due to its nutritional functions. Collagen is

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the most important structural biomacromolecule in sea cucumber body wall, and is highly

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related to the textual properties and food quality of sea cucumber. In this study, the types

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of constituent collagens of sea cucumber collagen fibrils were investigated, employing a

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commercially important species Apostichopus japonicus as the material. Proteomics and

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bioinformatics analysis revealed that collagen fibrils of A. japonicas are heterotypic. Two

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clade A and one clade B fibrillar collagens and two FACIT collagens were identified from

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the fibrils. Besides, the heterogeneity was also revealed in the pepsin-solubilized collagen

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(PSC) of A. japonicus by using the proteomics strategy. It implied that the previous

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conclusions on the type of sea cucumber collagen deduced from SDS-PAGE analysis

29

should be rechecked. The results provided novel insight into the composition of sea

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cucumber collagen fibrils.

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Keywords

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Sea cucumber; Collagen; Collagen fibrils; Pepsin-solubilized collagen; Proteomics

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1. Introduction

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Sea cucumbers are invertebrates of the class Holothuroidea in the phylum

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Echinodermata, and are consumed as traditional food materials in China, Japan, Korea, and

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some Southeast Asian countries (Li & Li, 2010). Due to their nutritional and bioactive

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functions, sea cucumbers have received increasing attention in recent years. The body wall

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is the main edible part of sea cucumbers, and collagens are the major biomacromolecules

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of the sea cucumber body wall (Saito, Kunisaki, Urano & Kimura, 2002), which account

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for about 70% of the total protein. It has been verified and acknowledged that collagens

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play critical roles in the food quality, especially the textural properties, of sea cucumbers

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and their processing products. And most of the previous studies on sea cucumber autolysis

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and sea cucumber processing (including boiling, drying, soaking and rehydration)

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attributed the change in the food properties of sea cucumber to collagen. Besides, sea

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cucumber collagens demonstrate favorable gelling properties and various bioactivities, and

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thus considered as promising additives and functional food ingredients (Bordbar, Anwar

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& Saari, 2011; Zhang et al., 2017).

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Collagens superfamily is composed of at least 28 different collagen types that consist of

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more than 40 distinct polypeptide chains (α chains) (Theocharis, Skandalis, Gialeli &

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Karamanos, 2016). It is well known that different types of collagens are distinct in structure,

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physical properties, tissue distribution and functions, as revealed in numerous publications

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(Bornstein & Sage, 1980; Ricard-Blum, 2011). The knowledge on the chemical

53

characteristics of collagen is the fundamental information for understanding the properties

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of collagen-rich food materials, including sea cucumbers. Sea cucumber collagen attracted

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increasing interests of food science researchers due to its significance to food properties of

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sea cucumber, and a few studies have investigated the type of collagens in sea cucumber

57

(Trotter, LyonsLevy, Thurmond & Koob, 1995; Saito et al., 2002; Cui et al., 2007; Liu,

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Oliveira & Su, 2010; Dong et al., 2011; Abedin et al., 2013; Siddiqui, Arief, Yusoff, Suzina

59

& Abdullah, 2013; Adibzadeh, Aminzadeh, Jamili, Karkhane & Farrokhi, 2014; Zhong,

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Chen, Hu & Ren, 2015). In those previous studies, pepsin-solubilized collagen (PSC) was

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widely employed as the material. The type of collagen in PSC was determined according

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to the results of SDS-PAGE analysis, i.e., the number of bands on the gel and their

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molecular weight. The previous reports consistently indicated that the collagen type of PSC

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is type I or type I-like (Supplementary Data 1), although some contradictions exist in the

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subunit composition.

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Fibrillar collagen can form multiple hierarchical structures, namely collagen α-chains,

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tropocollagen, collagen fibrils, and collagen fibers, in a bottom-up sequence (Ottani,

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Martini, Franchi, Ruggeri & Raspanti, 2002). PSC is the partially degraded product of

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collagen fibrils, in which the telopeptide regions are cleaved by pepsin while the triple

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helical structure remains intact (Zhu et al., 2012; Abedin et al., 2013). Compared with PSC,

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sea cucumber collagen fibrils have not been extensively investigated, and there was no

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clear information on the type of constituent collagens in the fibrils. Meanwhile, our recent

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study confirmed that fucosylated chondroitin sulfate was covalently attached to collagen

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fibrils in the sea cucumber body wall (Wang, Chang, Wu, Xu & Xue, 2018). To the best

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of our knowledge, no fibrillar collagens including type I collagen have been found to bear

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glycosaminoglycan chains (Jean Yves, Ulrich, Caroline & Claire, 2010). The presence of

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fucosylated chondroitin sulfate implies that heterogeneity might exist in the collagen

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composition of sea cucumber collagen fibrils. The aim of this study was to reveal the type

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of collagen molecules in the collagen fibrils of sea cucumber, using the proteomics

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technique instead of traditional SDS-PAGE analysis. Sea cucumber Apostichopus

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japonicus (synonym: Stichopus japonicus) was employed as the material due to its great

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commercial importance (FAO, 2016).

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2. Materials and methods

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2.1. Preparation of collagen fibrils

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Sea cucumber A. japonicus was purchased at a local aquatic market (Qingdao, China) in

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April 2017. Collagen fibrils were extracted from the body wall of A. japonicus at 4 °C

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according to a previously described method (Trotter et al., 1995). The body wall of A.

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japonicus was dissected, fractionated and stirred in deionized water at 4 °C. The tissue was

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subsequently stirred in 4 mM Na2EDTA solution with 0.02% NaN3, 1% protease inhibitor

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cocktail, and 0.1 M Tris-HCl (pH 8.0) for 12 h. The treated tissue was then washed with

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deionized water and slowly stirred for a further 12 h in deionized water containing 0.02%

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NaN3 and 1% protease inhibitor cocktail. Finally, the cloudy solution was filtered to

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remove residual dermis and then centrifuged for 30 min at 10000 g to collect collagen

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fibrils.

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2.2. Preparation of pepsin-solubilized collagen and its SDS-PAGE analysis

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PSC was prepared using a widely-adopted protocol (Trotter et al., 1995; Saito et al.,

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2002; Cui et al., 2007) with slight modification. Briefly, the fibrils were successively

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treated with 3 M guanidine-HCl in 50 mM sodium acetate for 1 d, 50 mM dithiothreitol in

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50 mM sodium acetate for 1 d, 0.1 M NaOH for 2 d and finally digested by 0.5 M acetic

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acid containing pepsin for 3 d. The PSC preparation was analyzed by SDS-PAGE on 7.5%

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resolving gel and 4% stacking gel. Proteins on the gels were stained with Coomassie

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Brilliant Blue R-250.

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2.3. Proteomics analysis

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2.3.1 Protein digestion

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The collagen fibrils were treated by 0.05 M trichloroethyl phosphate, 55 mM methyl-

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methyl-thiomethyl sulfoxide (MMTS), 8 M urea, 0.1 M Tris-HCl, 0.5 M triethyl

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ammonium bicarbonate and 3 μg trypsin (Liu, Zhou, Zhao, Hua & He, 2016). The

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cartridges C18 (Empore™ standard density SPE Cartridges C18, Sigma) were used for

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desalination. The bands of SDS-PAGE were excised and successively treated by water, 50%

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methyl alcohol, 50% ammonium bicarbonate and acetonitrile; and after dehydration, the

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protein in dried bands was digested as above described.

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2.3.2 LC-MS/MS

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Peptides were dissolved in 0.1% formic acid and 2% acetonitrile. LC–MS/MS was

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performed on a Q Exactive (Thermo Fisher Scientific, San Jose, CA, USA) equipped with

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the Ultimate 3000 RSLCnano Ultra Performance Liquid Chromatography (UPLC) system

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(Thermo Fisher Scientific, San Jose, CA, USA). The peptides were trapped on a reversed

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Acclaim PepMap C18 columns (0.075mm i.d., 15 cm long, Thermo Fisher Scientific, San

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Jose, CA, USA) using the Ultimate 3000 RSLC nano UPLC system (Liu et al., 2016). The

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parameters for mass spectrometry analysis are listed as follows: electrospray voltage: 2.0

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kV; normalized collision energy setting: 27%; capillary temperature: 275 °C; precursor

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scan range: 350 to 1800 m/z at a resolution of 70000; MS/MS fragment scan range: > 100

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m/z at a resolution of 17500.

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2.3.3 Protein identification

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Data were searched against the predicted proteome of A. japonicus deposited in the

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NCBI database (Assembly: ASM275485v1; Bioprojects: PRJNA354676) (Zhang et al.,

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2017) using ProteinPilot software (version 5.0.1, SCIEX) with the Paragon algorithm. The

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search parameters were as follows: enzyme, trypsin; cysteine alkylation, MMTS; false

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discovery analysis, yes; confidence score, > 0.05.

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2.4. Bioinformatics analysis

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The presence of the characteristic domain of collagen, i.e., collagen triple helix repeat

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(Interpro accession: IPR008160) in protein sequences was predicted by utilizing the

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software Interproscan (version: 5.35-74.0) (Jones et al., 2014). Multiple sequence

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alignments were implemented using ClustalX (Thompson, Gibson, Plewniak, Jeanmougin

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& Higgins, 1997), and the phylogenetic tree was subsequently constructed with MEGA6

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based on the neighbor-joining algorithm (Tamura, Stecher, Peterson, Filipski & Kumar,

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2013).

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3. Results and discussion

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3.1. Investigation on Collagen fibrils

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In the recently published high-quality genome of A. japonicas (Zhang et al., 2017), there

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are 56 sequences annotated as collagens using the BLAST algorithm. To improve the

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accuracy of the bioinformatic prediction, the 56 sequences were further analyzed in this

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study by the software package Interproscan that calculates utilizing the HMMER algorithm.

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As a result, 21 sequences were identified containing the characteristic protein domain of

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collagen (Supplementary Data 2). Furthermore, 5 of those sequences were confirmed in

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collagen fibrils by using the proteomics analysis (Table 1, Supplementary Table 1 and

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Supplementary Data 3).

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A phylogenetic tree was established from PIK60691.1, PIK60696.1 and PIK62545.1,

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with the experimentally characterized fibrillar collagen sequences deposited in the

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Swissprot database and collagen sequences of sea urchins and sponges (Fig. 1). The same

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type of collagen sequences located in the identical branch with robust bootstrap values,

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indicating that the phylogenetic tree is well constructed. The sea cucumber collagen

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sequences and sea urchin sequences are closely located as expected since sea cucumbers

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and sea urchins are both belonged to the phylum Echinodermata and close in the phylogeny.

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Nevertheless, none of the sea cucumber collagen sequences were included in branches

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of typical collagens. Comparing with vertebrate collagens, invertebrate collagens are

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relatively primitive and often fail to be classified into canonical collagen types which are

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defined based on the knowledge of vertebrate collagens (Zhang et al., 2007; Jean Yves et

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al., 2010). According to their evolutionary relationships, fibrillar collagen α chains are

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divided into three clades: clade A includes α1(I), α2(I), α1(II), α1(III) and α2(V) chains;

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clade B includes α1(V), α3(V), α1(XI) and α2(XI) chains; and clade C includes α1(XXIV)

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and α1(XXVII) chains (Zhang et al., 2007). The three clades were distinctly clustered in

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the phylogenetic tree (Fig. 1). The 1α, 5α and 2α chains of sea urchin located in the branch

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of clade A sequences, and the 6α chain of sea urchin located in the branch of clade B

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sequences, which was in accordance with previous studies (Jean Yves et al., 2010).

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According to the position of the sea cucumber collagen sequences in the phylogenetic tree,

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it could be confirmed that PIK60696.1 and PIK60691.1 are clade A fibrillar collagens

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while PIK62545.1 is clade B fibrillar collagens, as their sea urchin homologs.

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PIK55424.1 and PIK55422.1 could not be included in the phylogenetic tree of fibrillar

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collagen sequences (data not shown). Those two sequences are recorded as type IX

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collagens in the NCBI database. According to the common domain homology and

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functions, collagens can be classified into seven categories, i.e., fibrillar collagens, fibril-

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associated collagens with interrupted triple helices (FACITs), membrane-associated

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collagens with interrupted triple helices, network-forming collagens, anchoring fibrils,

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beaded filament-forming collagens, and multiple triple-helix domains collagens and

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interruptions (Theocharis et al., 2016). The type IX collagens are FACITs and usually

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present in the surface of cartilage collagen fibrils which are mainly composed of type II

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(Clade A fibrillar collagens) and type XI collagens (Clade B fibrillar collagens) (Olsen,

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1997). Intriguingly, it has been reported that the α2(IX) chains are covalently linked with

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glycosaminoglycan (McCormick, van der Rest, Goodship, Lozano, Ninomiya & Olsen,

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1987). It suggested that the fucosylated chondroitin sulfate might be attached to sea

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cucumber collagen fibrils via the type IX-like collagens. However, it should be mentioned

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that type IX collagens consist of three different α chains whereas only two homologous

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sequences can be found in the current genome assembly of A. japonicus; and the attachment

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sites of fucosylated chondroitin sulfate on the collagen chains are also unclear, which is

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under investigation in our lab.

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The results of proteomics and bioinformatics analysis confirmed that various types of

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collagens including clade A and clade B fibrillar collagens and FACIT collagens are

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present in the collagen fibrils of A. japonicas. In conclusion, the collagen fibrils of sea

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cucumber A. japonicas are heterotypic. Heterotypic collagen fibrils are widespread in

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vertebrates. For instances, human dermis collagen fibrils are composed of type I and III

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collagens (Fleischmajer, Macdonald, Perlish, Burgeson & Fisher, 1990), chicken cornea

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contains collagen fibrils consist of type I and V collagens (Birk, 2001), and human articular

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cartilage fibrils include type II and III collagens (Young, Lawrence, Duance, Aigner &

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Monaghan, 2000). The presence of heterotypic collagen fibrils has also been observed in

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invertebrates. Collagen fibrils from the dermis of cuttlefish Sepia officinalis are composed

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of type I-like and type V-like collagens (Bairati & Gioria, 2004). The 1α, 2α and 5α chains

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existed in sea urchin paracentrotus lividus collagen fibrils are supposed playing a similar

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structural function to that of the vertebrate types V/XI collagens in heterotypic fibrils

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(Cluzel, Lethias, Garrone & Exposito, 2004).

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The current study is the first effort to investigate the molecule composition of sea

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cucumber collagen fibrils using the proteomic strategy. As a result, the heterogeneity in the

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collagen fibrils of sea cucumbers is revealed for the first time. The observation is

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unexpected since all previous studies consistently concluded that PSC consists of type I or

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type I-like collagen α chains, i.e., no heterogeneity in the collagen type of sea cucumber

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collagen fibrils or its partially degraded products has ever been noticed. Since different

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types collagens are different in their thermal characteristics and mechanical characteristics

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(Bornstein et al., 1980; Ottani, Raspanti & Ruggeri, 2001), the observation suggested that

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enough attention should be paid to the heterogeneity and complexity of collagen in the

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mechanism study on sea cucumber food properties and their change in processing.

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Inspired by the above result, we were curious about whether the heterogeneity is also

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present in PSC. To answer the question, A. japonicas PSC was prepared and analyzed using

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SDS-PAGE as in previous studies, and comparatively investigated using the proteomic

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technique.

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3.2. Investigation on the PSC

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The SDS-PAGE is widely adopted in the determination of collagen type. The collagen

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type was concluded majorly based on the number of protein bands on the gel and their

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molecular weight, assuming that each band represents a single collagen α chain. The SDS-

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PAGE image of the A. japonicas PSC was shown in Fig. 2. It is worth noting that previous

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reports on A. japonicas PSC showed different SDS-PAGE band patterns. Saito (2002)

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observed two bands on the SDS-PAGE gel, and the subunit composition of the PSC was

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therefore deduced as (α1)2α2; whereas Cui (2007) and Dong (2011) concluded that the

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subunit composition of the PSC is (α1)3 since only one SDS-PAGE band appeared. In the

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current study, the PSC exhibited two clear and discrete bands (Fig. 2), which resembled

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the result of Saito (2002). The molecule weight of band 1 and band 2 was estimated to be

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146 kDa and 136 kDa respectively.

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Although the PSC showed a typical SDS-PAGE image, proteomics identification of

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band 1 and band 2 demonstrated that all the bands contained several collagen α chains

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(Table 1) rather than a single one. Especially, the band 1 contained two clade A collagens

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(PIK60691.1 and PIK60696.1) and one clade B collagen (PIK62545.1), which confirmed

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that PSC is heterotypic. PIK60691.1 and PIK60696.1 were found in both band 1 and band

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2, which might be attributed to that the collagen α chains were degraded to varying degrees

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by pepsin, or caused by mRNA alternative splicing events. The two FACIT collagens

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(PIK55424.1 and PIK55422.1) were not detected in the PSC, which might be susceptible

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to the NaOH and pepsin treatments, due to the β-elimination of glycosaminoglycan chains

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and their outermost position on the surface of collagen fibrils.

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The investigation on PSC intriguingly revealed: 1) A. japonicas PSC is heterotypic, and

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the previous conclusions on the collagen type of sea cucumber PSC should be rechecked;

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2) one SDS-PAGE band of sea cucumber PSC might not just contain one collagen α chain

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as the assumption, indicating that SDS-PAGE is not a reliable strategy for determining the

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collagen type; 3) proteomics can provide more detailed and accurate information for the

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determination of collagen type.

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4. Conclusions

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The study revealed that collagen fibrils of sea cucumber A. japonicas are heterotypic.

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Collagen sequences identified from the fibrils included two clade A fibrillar collagens, one

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clade B fibrillar collagen, and two FACIT collagens. Pepsin-solubilized collagen of A.

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japonicas is also heterotypic rather than type I or type I-like as previously reported. It

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indicated that the conclusions on collagen type deduced from SDS-PAGE analysis should

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be treated with caution. It is the first report on the heterogeneity existed in sea cucumber

249

collagen fibrils. The present study provided novel insight into the composition of sea

250

cucumber collagen fibrils, implying that the heterogeneity and complexity of collagen

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should be taken into account in future studies on food properties and processing of sea

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cucumber.

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Conflict of interest The authors declare no competing financial interest.

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Acknowledgments

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This work was supported by the Fundamental Research Funds for the Central

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Universities (201941005), and the National Natural Science Foundation of China

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(31671883).

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Figure Captions

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Fig. 1. Phylogenetic analysis of identified fibrillar collagen sequences in A. japonicus

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collagen fibril with previously reported sequences. All reviewed fibrillar collagens in the

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Swissprot database and sea urchin fibrillar collagens were involved, and the sequence of

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sponge which was considered as the most primitive fibrillar collagen was employed as the

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root. The database accession and organism species (in brackets) were successively listed

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in labels, and branches of typical fibrillar collagens were compressed for the conciseness

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(the detailed information of sequences was shown in Supplementary Data 4)

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Fig. 2. SDS-PAGE of the PSC. The indicated band 1 and band 2 were excised out for the

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further proteomics analysis.

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