Journal of Applied Glycoscience
Online ISSN : 1880-7291
Print ISSN : 1344-7882
ISSN-L : 1344-7882
Regular Papers
Characterization of Proteoglycan and Hyaluronan in Water-based Delipidated Powder of Salmon Cartilage
Ikuko KakizakiAyako MiuraSeiko ItoTakashi MinetaHong Jin SeoYoji Kato
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2015 Volume 62 Issue 3 Pages 115-120

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Abstract

Salmon cartilage proteoglycan fractions have attracted attention as new ingredients of functional food and cosmetics. We recently developed methods for water-based delipidation and powderization for preservation of salmon head cartilage. In this study, global molecular images of proteoglycan in the water-based delipidated powder were analyzed by biochemical techniques and atomic force microscopy. Proteoglycans in this powder maintained their whole native structures including core proteins and glycosaminoglycans. Hyaluronan contained in this powder showed a distribution with high molecular weight like native hyaluronan in cartilage. Analytical data presented here provides assurance of the quality of the proteoglycan powder obtained using these methods.

Abbreviations

PG, proteoglycan; EGF, epidermal growth factor; GAG, glycosaminoglycan; GlcUA, glucuronic acid; Gal, galactose; Xyl, xylose; ChS, chondroitin sulfate; HA, hyaluronan; KS, keratan sulfate; G1 domain, globular domain 1; G3 domain, globular domain 3; PGNP, proteoglycan natural powder; GdnHCl, guanidine hydrochloride; HABP, HA binding protein; PA, 2-pyridylamine; AFM, atomic force microscopy.

INTRODUCTION

Salmon cartilage proteoglycan (PG) fractions are expected to be useful as ingredients of functional foods and cosmetics, and possibly have promise as ingredients of pharmaceutical products or tissue-engineered medical products. Functions of PG fractions such as immunomodulating effect or epidermal growth factor (EGF)-like effect on cultured cells and therapeutic properties for colitis or osteoporosis in animal experimental models have been reported.1) 2) 3) 4) 5) 6) 7) 8) 9) 10) In PG preparation for industrial uses, frozen salmon heads are thawed. During long term frozen storage, not only does oxidation of the fat occur, but also difficulties in ensuring storage space are encountered. To alleviate these problems, we established methods for water-based delipidation and powderization for preservation of salmon head cartilage11). Safety studies of this powder confirmed its suitability as a food ingredient.12) Functional studies of PG fractions from this powder also revealed dermatological efficacy.13) 14)

PGs are glycoconjugates composed of one or multiple glycosaminoglycans (GAGs) covalently linked to the core protein through the common glucuronic acid-galactose-galactose-xylose-serine (GluUAβ1-3Galβ1-3Galβ1-4Xylβ-O-Ser) linkages, in the case of PGs that carry chondroitin sulfate (ChS)/dermatan sulfate or heparin/heparan sulfate chains.15) The prominent PG in mammalian cartilage is aggrecan. Recently, we demonstrated the prominent PG in salmon cartilage to also be aggrecan and other PG in salmon cartilage to be present in only negligible trace amounts, and we characterized the global molecular structure of purified salmon aggrecan.16) 17) In this study, we analyzed aggrecan (henceforth PG) and hyaluronan (HA) in the water-based delipidated powder of salmon nasal cartilage in order to elucidate their structural characteristics, quantity and size, respectively.

MATERIALS AND METHODS

Materials. Frozen salmon heads were purchased from a local agency in Aomori, Japan. Bovine nasal cartilage was obtained from a local slaughtering center in Aomori, Japan, following official procedures. DEAE-Sephacel and Sepharose CL-4B were from GE Healthcare Japan Corp. (Tokyo, Japan). Mouse monoclonal antibody, 12/21/1-C-6 (recognizing G1 domain of rat chondrosarcoma PG), and antirabbit polyclonal antibody against the synthetic peptide (2277DGHPMQFENWRPNQPDN2293) in the human aggrecan G3 domain, were from the Developmental Studies Hybridoma Bank of the University of Iowa (USA) and Affinity BioReagents Inc. (Golden, USA), respectively. Anti-salmon aggrecan EGF was generated by immunizing rabbits with synthetic peptide (1069RDLCEPNQCGTGTCSVQDGI1088) of an EGF-like module of salmon nasal cartilage aggrecan (Immuno-Biological Laboratories Co., Ltd., Gunma, Japan).17) Peroxidase-conjugated rabbit anti-mouse immunoglobulins and peroxidase-conjugated goat anti-rabbit immunoglobulins were from Dako Japan Inc. (Tokyo, Japan). Actinase E (protease from Streptomyces griseus) and cellulase (from Aspergillus niger) was from Kaken Pharmaceutical Co. (Tokyo, Japan) and Sigma-Aldrich Corporation (St. Louis, USA), respectively. Chondroitin ABC lyase (from Proteus vulgaris, EC 4.2.2.4), HA lyase (from Streptomyces hyalurolyticus, EC 4.2.2.1), and unsaturated disaccharide standards were from Seikagaku Biobusiness Co. (Tokyo, Japan). Other analytical grade reagents were obtained from commercial sources.

Preparation of water-based delipidated powder of salmon cartilage. Water-based delipidated powder of salmon cartilage was prepared according to the methods of the patent11). Briefly, cartilage was separated from the salmon head and washed with water. The cartilage was minced and suspended in 1‒2 volumes of water and centrifuged at 16,000 × G for 30 min at room temperature, then residues were collected. This procedure was repeated twice and the obtained residues were lyophilized and powdered using a centrifugal powdering machine. The powder was suspended in 10 volumes of ethanol, filtrated using filter paper (Toyo Roshi No. 2; Toyo Roshi Kaisha, Ltd., Tokyo, Japan), and dried to give water-based delipidated powder of salmon cartilage. In this paper, we refer to this powder as “PG natural powder (PGNP)”. Note that in this procedure only food use materials (water and ethanol) were used.

Extraction and purification of PG from the PGNP of salmon cartilage. Crude salmon cartilage PG was extracted from the PGNP by the common extraction procedure using 4M guanidine hydrochloride (GdnHCl).18) PG was then purified by DEAE-Sephacel chromatography followed by Sepharose CL-4B chromatography according to the procedure of our previous report.17) Extraction and purification was performed in the presence of protease inhibitors. Important points were as follows: Crude PG was extracted from 8.18 g of the PGNP. The extract was precipitated with ethanol and then dissolved in distilled water and re-precipitated to yield 9.46 g of lyophilized material, 1 g of which contained 122 mg uronic acid, and 12.5 mg protein. Ten mg (by uronic acid) of the extracted crude PGs were separated by DEAE-Sephacel column (1.8 × 15 cm) using eluents; 7 M urea in 50 mM Tris-HCl buffer (pH 7.4) with a linear gradient (0 to 1.0 M) of NaCl followed by 2.0 M of NaCl in the same buffer, at a flow rate of 0.4 mL/min, and 6.3 mL fractions were collected. Fractions positive both for uronic acid and protein, and whose elution position corresponded to around 0.5 M NaCl were pooled, desalted and concentrated by ultrafiltration to yield chondroitin sulfate-PG (Frs. 53‒77 as pool I). The pool I was further fractionated by Sepharose CL-4B column (1.8 × 110 cm) using 4 M GdnHCl in 50 mM sodium acetate buffer (pH 6.0) as an eluent at a flow rate of 0.33 mL/min. Fractions (3.3 mL) were collected and those positive for both uronic acid and protein were pooled (Frs. 26‒28 as pool II) and concentrated by ultrafiltration to yield purified PG (1.06 mg of uronic acid, 0.78 mg of protein).

Analytical methods. Uronic acid content was determined by the carbazole sulfuric acid method19) and protein content was determined by the method of Bradford20) or by monitoring the UV absorbance at 280 nm.

Size analysis of purified PGs and HA, and analysis of sulfation degree were performed using HPLC according to our previous report.17) 21)

For the unsaturated disaccharide composition analysis, GAG chains were liberated from PG by treatment with cellulase (from A. niger) by its endo-β-xylosidase activity.22) Prior to the cellulase treatment the core protein of PG was digested with actinase E. The unsaturated disaccharide compositions of GAGs were determined by HPLC after digestion with chondroitin ABC lyase.23) 24)

HA content was measured by ELISA-like assay using HA binding protein (HABP) according to the manufacturer’s instructions for HA Assay Kit (Seikagaku Biobusiness Co.). The absorbance at 490 nm (control wavelength, 630 nm) of each well was measured by a microplate spectrophotometer xMarkTM (Bio-Rad Laboratories KK., Tokyo, Japan). Size analysis of HA was performed according to our previous report.17) Briefly, fractions of 28‒37, that were eluted with about 0.2 M of the NaCl gradient, were recovered by DEAE-Sephacel chromatography. From our repeated experiments, these combined fractions would be expected to contain HA and were named “pool X”. The pool X was concentrated and fractionated by Shodex OHpak SB-804 HQ gel filtration column and HA content in each fraction (1.0 mL/fraction) was measured.

All enzymatic digestions for analytical experiments were performed exhaustively under optimal conditions and reactions stopped by boiling at 100°C for 3 min.

HPLC analyses. HPLC was performed on a Hitachi ELITE LaChrom system equipped with a model L-2420 UV-VIS detector and model L-2490 RI detector or model L-2485 fluorescence detector (Hitachi High-Technologies Corporation, Tokyo, Japan).

For size estimation, Shodex OHpak SB-805 HQ column (8.0 × 300 mm, Shodex, Showa Denko K.K., Kawasaki, Japan) for PGs and Shodex OHpak SB-804 HQ column (8.0 × 300 mm, Shodex, Showa Denko K.K.) for HA were used, both with 0.2 M NaCl as an eluent. Elution was carried out at 40°C at a flow rate of 1.0 mL/min and 0.5 mL/min, respectively. As size standards, pullulans (Mr = 78.8 × 104, 40.4 × 104, 21.2 × 104, 11.2 × 104, 4.73 × 104, 2.28 × 104, 1.18 × 104, and 0.59 × 104, Shodex, Showa Denko K.K.) were used for the analysis of PGs, and HAs (Mr = 190 × 104, 80 × 104, 30 × 104, 10 × 104, and 4.1 × 104, kindly supplied by Denki Kagaku Kogyo, Tokyo, Japan) were used for the analysis of HA. The eluate was monitored by UV absorbance and differential refractive index. Detailed procedures regarding pretreatment of PG before HPLC to analyze core protein and GAG chains were described in our previous report.17)

For the analysis of sulfation degree, ion exchange HPLC was performed on a TSKgel DEAE-5PW column (7.5 × 75 mm, Tosoh Corp., Tokyo, Japan) with a linear gradient of NaCl from 0 to 1.0 M for 60 min at a flow rate of 1.0 mL/min at 40oC.21) The pyridylaminated GAG (PA-GAG) chains were monitored by the fluorescence of 2-pyridylamine (PA) at excitation and emission wavelengths of 320 and 400 nm, respectively. The calibration curve was plotted by using PA-Ch4S standards desulfated for various times (0, 12, 24, 48, and 72 h),25) and whose disaccharide compositions were known by unsaturated disaccharide analysis.

For unsaturated disaccharide analysis, a YMC-Pack Polyamine II column (4.6 × 250 mm, YMC Co., Kyoto, Japan) was used. Disaccharides were eluted with a linear gradient of NaH2PO4 from 16 to 478 mM over 60 min at a flow rate of 1.0 mL/min at 30°C. The unsaturated disaccharides were monitored by UV absorbance at 232 nm.

Dotblot analysis using antibodies specific for domains of aggrecan. PG (0.2 μg of protein), which had been reduced-alkylated, was blotted to a PVDF membrane (Millipore Corporation, Billerica, USA) using a slot blotter (Scie-Plas Ltd., Cambridge, UK), and analyzed by probing with antibodies against aggrecan G1 domain, aggrecan G3 domain, or salmon aggrecan EGF-like module according to our previous report.17)

AFM imaging. Atomic force microscopy (AFM) imaging was performed according to our previous report17) following Yeh et al.26 and Ng et al.27) except with a Nanocute AFM system (SII NanoTechnology Inc., Tokyo, Japan) using SSS-NCH probes (Nano World AG, Neuchâtel, Switzerland).

RESULTS AND DISCUSSION

Purification of PG and HA from PGNP. PG and HA were purified from 4 M GdnHCl-extract of the PGNP by chromatographies (Fig. 1). Fractions 28‒37 (pool X in Fig. 1(A)) of DEAE column chromatography were collected as they were expected to contain HA. Combined fractions 53‒77 (pool I in Fig. 1(A)) of DEAE column chromatography were further fractionated by CL-4B column chromatography and the fractions 26‒28 (pool II) were collected as purified PG. Both chromatograms were similar to those of 4 M GdnHCl-extract from salmon cartilage reported previously,17) suggesting that both PGNP extract and cartilage extract contain PGs with similar electric charges and sizes based on their GAG moieties.

Fig. 1.

DEAE-Sephacel and Sepharose CL-4B column chromatography of PGNP extract.

The 4 M GdnHCl-extract of PGNP was fractionated by DEAE-Sephacel chromatography (A) followed by Sepharose CL-4B chromatography (B). Broken lines indicate the gradient curves of NaCl and arrows the specific NaCl concentrations. Solid circles, uronic acid content detected by carbazole sulfuric acid method; open circles, protein content detected by the method of Bradford (A) or protein content by monitoring the UV absorbance at 280 nm (B). Bars with Greek numerals indicate the fractions combined for recovery of material as Pools I, II, and X: pool I (Frs. 53‒77), pool II (Frs. 26‒28), and pool X (Frs. 28‒37). Pool I from DEAE column chromatography was further fractionated by Sepharose CL-4B column chromatography. Pool II from Sepharose CL-4B chromatography was recovered and analyzed. The material indicated as pool X was collected for the analysis of HA.

Molecular size distribution of purified PG from PGNP. Determining the absolute molecular size distribution of large PGs such as aggrecan, or even of its GAG moiety, is not possible because of the lack of appropriate size standards due to the heterogeneity of GAGs. However, we estimated the relative size distributions of PG in PGNP extract (sizes of whole molecules, core protein, and one GAG) by gel filtration HPLC using various sizes of pullulans as size standards (Fig. 2). Relative size distributions are shown in Table 1 and the calculated values from Table 1 are shown in Table 2. The tables also include the values of the PGs from cartilage extract for comparison. Numbers of GAG chains in the PGs were calculated as follows: The total Mr of the GAG chains was obtained by subtracting the estimated Mr of a core protein, (B) in Table 1, from the estimated Mr of the non-treated PG, (A) in Table 1. Numbers of GAG chains in the PGs were obtained by dividing the total Mr of carbohydrates by Mr of one GAG chain, (C) in Table 1. For example, the number of GAG chains in the PG purified from PGNP extract (pool II) was obtained by the following formula: (1,620,000‒94,000)/164,000 = 9.3. The relative size distributions and the calculated values were similar between PG in PGNP extract and PG in cartilage extract. The reason for the tiny differences between them may be dependent on the habitat environment and also due to the variations of salmon cartilage source (ages, sex), but are not related to the position in the head of the cartilage.28) And size-relationships of values between salmon and bovine PG shown here also support our previous report.17)

Fig. 2.

Gel filtration HPLC of PG purified from PGNP.

PGs were analyzed by Shodex OHpak SB-805 HQ gel filtration column before treatment (a) or after treatment with LiOH followed by chondroitin lyase ABC (b) or after treatment with actinase E (c). Solid arrows indicate the elution positions of size standards of pullulan with defined approximate molecular weight. Dashed arrows indicate the void volume (V0) and total column volume (Vt).

Table 1.

Molecular weight estimated by gel filtration HPLC. PG samples were analyzed with a Shodex OHpak SB-805 HQ column. Various sizes of pullulan were used as size standards.

PG, proteoglycan; PGNP, proteoglycan natural powder. aTreated with LiOH followed by chondroitin lyase ABC digestion. bDigested with Actinase E. Molecular weight of material eluting at the peak position.

Table 2.

Calculated values of glycosaminoglycan chains from Table 1.

PG, proteoglycan; PGNP, proteoglycan natural powder. Peak top data from standard size pullulans.

Sugar chain analyses of purified PG from PGNP. Degrees of total sulfation were determined as 0.86 per disaccharide unit in PG from PGNP extract by our method using TSKgel DEAE-5PW,21) and the value was similar to those of PGs in cartilage extract of salmon or bovine that we previously reported.17) Salmon aggrecan does not have a keratan sulfate (KS) attachment domain, which is common in mammalian aggrecans along with a ChS attachment domain.16) And, in fact, possible monosaccharide components of KS were detected in only trace amounts.17) Therefore, we performed unsaturated disaccharide composition analysis only after chondroitin ABC lyase for focusing chondroitin sulfate structure. The compositions of ChSs were similar between PG in PGNP extract and PG in salmon cartilage extract and differed from those of bovine PG (Table 3). This indicates that pretreatment process before extraction has no effect on the sugar chains if the cartilages are from the same species.

Table 3.

Unsaturated disaccharide compositions of GAG moiety of salmon nasal cartilage PG. Unsaturated disaccharide compositions of GAGs were determined by HPLC on a YMC-Pack Polyamine II column after digestion with chondroitin ABC lyase. The results are expressed as a percentage of each detected unsaturated disaccharide to total unsaturated disaccharides.

PG, proteoglycan; PGNP, proteoglycan natural powder. aΔDi-0S, ΔGlcUA-β1-3GalNAc; ΔDi-4S, ΔGlcUA β1-3GalNAc(4S); ΔDi-6S, ΔGlcUAβ1-3GalNAc(6S); ΔDi-diSD, ΔGlcUA(2S) β1-3GalNAc(6S); 2S, 4S, and 6S, represent 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate. bnot detected.

Immunological analysis of purified PG from PGNP. Dot blot analysis using antibodies against the G1, G3 domains or EGF-like module of salmon cartilage PG showed that PG in PGNP extract has all of these domain structures (Fig. 3) as well as PG in cartilage extract. This indicates that the preparation procedure of PGNP does not affect the core protein structure.

Fig. 3.

Immunological analysis of PG in PGNP.

(A) Domain structure of salmon cartilage aggrecan core protein. G1, globular domain 1; IGD, interglobular domain; G2, globular domain 2; GAG, glycosaminoglycan attachment domain; EGF, EGF-like module, G3, globular domain 3. (B) Dot-blotted proteins (0.2 μg each), which had been reduced-alkylated, were probed with antibodies to aggrecan G1 domain, G3 domain, or salmon aggrecan EGF-like module. 1, 4 M GdnHCl-extract of salmon nasal cartilage; 2, PG purified from 4 M GdnHCl-extract of salmon nasal cartilage; 3, 4 M GdnHCl-extract of PGNP; 4, PG purified from 4 M GdnHCl-extract of PGNP; 5, PG purified from 4 M GdnHCl-extract of bovine nasal cartilage.

Molecular images of PGs. We observed the molecular images of PG monomers in PGNP extract by AFM and found them to be similar to those of PG monomers in salmon cartilage extract that we previously reported17) (Fig. 4(A)). Also, the images were different from those of PG purified from bovine nasal cartilage extract as a control (Fig. 4(C)). The core proteins of salmon PGs were shorter and have a lower number of GAG chains, however, GAGs of salmon PG were longer than those of bovine, supporting our previous data.17) As well as the above described analytical data, AFM images suggested that PG in PGNP extract is equal in quality to PG in salmon cartilage extract.

Fig. 4.

AFM images of PG purified from PGNP.

Purified PG from PGNP was analyzed by AFM. Height image (A) and amplitude image (B) of PG purified from 4 M GdnHCl-extract of PGNP (pool II in Fig. 1(B)); height image (C) and amplitude image (D) of PG purified from 4 M GdnHCl-extract of bovine nasal cartilage. Magnification bar represents 200 nm. Color bars under the height image and amplitude image indicate ranges of height and error signal of amplitude change in the imaged area, respectively.

Content and size distribution of HA in extract from PGNP. PGs show functions with interacting HA with high molecular weight or other molecules, rather than PG monomers independently.15) 29) 30) Therefore, as materials for future applications in various fields, materials containing both HA, PG, and related molecules would be expected to be more valuable. In order to determine whether the PGNP-extract contains HA, which would interact with PG, combined fractions 28‒37 (pool X) were concentrated and HA content was measured. The HA content in the PGNP-extract was 1.6 μg per 1 mg total uronic acid, thus, the ratio of HA (by ELISA like assay) to uronic acid (by carbazole sulfuric acid method) is 1: 625. To determine the size distribution of HA, the above sample was fractionated by gel filtration HPLC using a Shodex OHpak SB-804 HQ column and the HA content of the fractions measured (Fig. 5). Size distribution of HA in the PGNP-extract was found to be broad, from 2,000 to 2,400,000 with the peak at 100,000, which was similar to that of HA in cartilage-extract of salmon and bovine as reported previously.17) PGNP is thought to be a valuable material because PGNP contains HA with high molecular weight.

Fig. 5.

Gel filtration HPLC of HA contained in PGNP extract.

Pool X (combined fractions indicated in DEAE chromatogram) (Fig. 1(A)) was concentrated and fractionated by Shodex OHpak SB-804 HQ gel filtration column (8.0 × 300 mm). Fractions (1.0 mL/fraction) were collected and HA content in each fraction was measured. Solid circles, pool X without HA lyase (from S. hyalurolyticus) treatment; open circles, pool X treated with HA lyase (from S. hyalurolyticus). Arrows indicate the elution positions of size standards of HA. Dashed arrows indicate the void volume (V0) and total column volume (Vt).

In conclusion, we elucidated the existence of intact PG and HA both with high molecular weight in salmon PGNP. Our preparation procedure of PGNP did not affect the quality of PG and HA, thus, obtained molecules reflect native forms as monomers in cartilage. This indicates that not only does this preparation of PGNP alleviate problems during long term storage of salmon heads but also that it leads to efficient extraction of high molecular weight PG and HA as a second step.

ACKNOWLEDGEMENTS

This work was supported by the Regional Innovation Strategy Support Program (City Area Type) and Grants-in-Aid for Scientific Research (Nos. 23570157, 26462470 for I. K., and 23650253 for T. M.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

REFERRENCES
 
© 2015 by The Japanese Society of Applied Glycoscience
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