In Vitro Synthesis of Glycogen: the Structure, Properties, and Physiological Function of Enzymatically-Synthesized Glycogen

Biologia 66/3: 387—394, 2011 Section Cellular and Molecular Biology DOI: 10.2478/s11756-011-0053-y Review

In vitro synthesis of glycogen: the structure, properties, and physiological function of enzymatically-synthesized glycogen

Hideki Kajiura1,2,HirokiTakata 2, Tsunehisa Akiyama2,RyoKakutani2, Takashi Furuyashiki2,IwaoKojima2, Toshiaki Harui1 & Takashi Kuriki2

1Development Department, Food Material Division, Glico Foods Co., Ltd., 7–16 Kasuga-cho, Takatsuki, Osaka 569-0053, Japan; e-mail: [email protected] 2Institute of Health Sciences, Ezaki Glico Co., Ltd., 4-5-6, Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan

Abstract: This review describes a new enzymatic method for in vitro glycogen synthesis and its structure and properties. In this method, short-chain amylose is used as the substrate for branching enzymes (BE, EC 2.4.1.18). Although a kidney bean BE and Bacillus cereus BE could not synthesize high-molecular weight glucan, BEs from 6 other bacterial sources produced enzymatically synthesized glycogen (ESG). The BE from Aquifex aeolicus was the most suitable for the production of glycogen with a weight-average molecular weight (Mw) of 3,000–30,000 k. The molecular weight of the ESG is controllable by changing the concentration of the substrate amylose. Furthermore, the addition of amylomaltase (AM, EC 2.4.1.25) significantly enhanced the efficiency of this process, and the yield of ESG reached approximately 65%. Typical preparations of ESG obtained by this method were subjected to structural analyses. The average chain length, interior chain length, and exterior chain length of the ESGs were 8.2–11.6, 2.0–3.3, and 4.2–7.6, respectively. Transmission electron microscopy and intrinsic viscosity measurement showed that the ESG molecules formed spherical particles. Unlike starch, the ESGs were barely degraded by pullulanase. Solutions of ESG were opalescent (milky-white and slightly bluish), and gave a reddish- brown color on the addition of iodine. These analyses revealed that ESG shares similar molecular shapes and solution properties with natural-source glycogen. Moreover, ESG had macrophage-stimulating activity and its activity depends on the molecular weight of ESG. We successfully achieved large scale production of ESG. ESG could lead to new industrial applications, such as in the food, chemical, and pharmaceutical fields. Key words: glycogen; branching enzyme; enzymatic synthesis; α-glucan; polysaccharide; structure. Abbreviations: AM, amylomaltase; BE, branching enzyme; DP, degree of polymerization; ESG, enzymatically synthesized glycogen; GP, α-glucan phosphorylase; HPSEC-MALLS-RI, high-performance size-exclusion chromatography with a multi- angle laser light scattering photometer and a differential refractive index detector; IAM, isoamylase; NSG, natural-source glycogen; SP, sucrose phosphorylase; TEM, transmission electron microscopy.

Glycogen has also been isolated from several higher plants. In animal tissues, glycogen is synthesized from UDP-glucose Glycogen is the storage form of glucose in animals, via the cooperative action of glycogenin (EC 2.4.1.186), fungi, bacteria, yeasts, and archaea. The polysaccharide glycogen synthase (EC 2.4.1.11), and branching en- is a highly branched (1→4)(1→6)-linked α-d-glucan zyme (BE, 1,4-α-d-glucan:1,4-α-d-glucan 6-α-d-(1,4-α- with a high molecular weight (106–109) (Geddes 1986). glucano)-transferase, EC 2.4.1.18). Based on the results of electron microscopy, it has been Glycogenisolatedfromanimal tissues or shellfish shown that glycogen consists of spherical particles with has been used to stimulate the exudation of phagocytic diameters of 20–40 nm (β-particles), and that the β- cells into the peritoneal cavities of experimental ani- particles often associate into much larger α-particles mals in immunological studies (Thorpe & Marcus 1967; (∼200 nm). The molecular weight of an individual Mantovani 1981). It has also been reported that glyco- β-particle has been shown to be approximately 107 gen has an antitumor effect, probably through its im- (Geddes 1986). Glycogen aqueous solution is opalescent munomodulating activity, and that this activity seems (milky-white and slightly bluish), and gives a reddish- to depend on the fine structure of glycogen (Takaya et brown color on the addition of iodine. Amylopectin, a al. 1998; Takaya 2000). Glycogen has long been consid- major component of starch, is also a highly branched ered to have health benefits as a food ingredient. As re- (1→4)(1→6)-linked α-d-glucan. However, the degree of vealed below, we demonstrated that enzymatically syn- branching, namely the number of (1→6)-α-d-glucosidic thesized glycogen (ESG) has a stimulating activity on linkages, in glycogen is about twice that in amylopectin. immunocompetent cells, such as macrophages. Glyco- A glycogen-type polymer, referred to as phytoglycogen, gen has also been used as a raw material in the cosmetic

c 2011 Institute of Molecular Biology, Slovak Academy of Sciences 388 H. Kajiura et al. industry and as a carrier to enhance the yield of DNA vivo synthesis of glycogen by glycogen synthase and BE. during precipitation with organic solvent (Heath et al. Then, a question has come up. Can we synthesize glyco- 2001). gen simply using amylose as a substrate? The published results to date have all been negative: BE can synthe- In vitro synthesis of glycogen size branched glucans but their molecular weights are much lower than that of glycogen. For example, Praznik Extraction of glycogen from natural resources such as et al. (1992) reported that the action of potato BE on animal tissues or shellfishes is laborious and costly pro- amylose with a weight-average molecular weight (Mw) cess. Therefore, in vitro synthesis may be possible an- of 67.6 k resulted in a branched product with a Mw of swer to obtain glycogen in industrial scale. Actually, 33.5 k. Boyer et al. (1982) analyzed the action of maize the in vitro synthesis of glycogen has been success- BE isozyme I by gel filtration chromatography and ob- fully achieved by the combined action of α-glucan phos- served that the product was eluted more slowly than the phorylase (GP, EC 2.4.1.1) and BE using glucose-1- substrate. Kitamura (1996), using light-scattering anal- phosphate (G-1-P) as a substrate (GP-BE method) yses, also reported that potato BE brought about a de- about 70 years ago. (Cori & Cori 1943). At that time, crease in the molecular weight of the substrate amylose. enzymes were extracted from animal tissues and costly. We have reported that Bacillus cereus BE produced Additionally, GP from animal tissues is allosteric en- branched glucan with a Mw of 50 k from amylose with zyme and need to be phosphorylated or added an acti- Mw of 320 k (Takata et al. 2005). The decreases in the vator (glucose-6-phosphate) (Fukui et al. 1982). How- molecular weight of the substrate can be attributable to ever, microbial or plant enzymes have been developed a cyclization reaction by BE; it has been demonstrated andusedforthein vitro synthesis by many researchers that BE catalyzes a cyclization reaction in addition to (e.g., Tolmasky & Krisman 1987; Fujii et al. 2003; a branching reaction in vitro. As a result of cycliza- van der Vlist et al. 2008). The ESG produced by tion, the molecular weight of the substrate should be the GP-BE method has properties similar to those of decreased. By contrast, an inter-chain branching reac- the natural-source glycogen (NSG) (Kitahata & Okada tion results in both larger and smaller molecules if two 1988). UDP-glucose, a natural substrate for in vivo molecules of the same size are used as a substrate. An glycogen synthesis, was also used as substrate by us- intra-chain branching reaction should not change the ing glycogen synthase and BE as catalysts (Parodi et molecular weight. However, most studies have used BE al. 1969). Although both G-1-P and UDP-glucose are from plant sources and relatively long-chain amyloses expensive for industrial applications, the former could were used as the substrate. To evaluate the possibility be obtained from the inexpensive substrate sucrose by of glycogen synthesis from amylose, we tested the ac- using sucrose phosphorylase (SP, EC 2.4.1.7) (SP-GP- tivity of eight types of BE from various sources using BE method) (Ohdan et al. 2006). Furthermore, we have relatively short-chain amyloses as the substrates. We demonstrated that ESG produced by the SP-GP-BE found that some BEs from bacterial sources can syn- method has immunomodulating activity (Ryoyama et thesize glycogen (ESG). We then compared some of the al. 2004). However, the yield of glycogen from sucrose properties of the ESG with those of NSG. Furthermore, was less than 40%, and a more efficient method is re- we found that ESG has physiological function. quired to meet the demand for glycogen for industrial applications. Actions of BEs from several sources on amyloses In the GP-BE and SP-GP-BE methods, GP elon- gates a chain of α-(1→4)-glucan and BE introduces To evaluate the possibility of producing glycogen from branch points in the growing chains, mimicking the in amylose, we tested the actions of BEs from eight sources

Table 1. Weight-average molecular weight and yield of α-glucans produced by the action of several BEs on two amyloses.a

Substrate Source of BE Amylose A Amylose AS10

Mw (k) Yield (%) Mw (k) Yield (%)

kidney bean N.D.b N.D. 185 16.0 Bacillus cereus N.D. N.D. 87 25.5 Escherichia coli 3600 3.9 3900 25.2 Bacillus caldovelox 5910 4.5 10200 23.7 Bacillus caldolyticus 36300 1.2 30400 5.3 Bacillus stearothermophilus 2620 6.7 5760 32.0 Aquifex aeolicus 9370 10.1 20400 59.0 Rhodothermus obamensis 10300 31.2 45300 66.7 a A reaction mixture (1 mL) containing 5 mg of Amylose A or Amylose AS-10, 50 mM buffer (optimum pH) and 200 U of various BEs was incubated at optimum temperature for each enzyme for 24 h. b Not detected by HPSEC-MALLS-RI. In vitro synthesis of glycogen 389 on short-chain amyloses using high-performance size- exclusion chromatography with a multi-angle laser light scattering photometer and a differential refractive index detector (HPSEC-MALLS-RI) (Kajiura et al. 2008). The substrates used were Amylose A (Mn,number- average molecular weight, 2.9 k; Nacalai Tesque, Inc., Kyoto, Japan) and Amylose AS-10 (Mw,10k;Nakano Vinegar Co., Ltd., Aichi, Japan). In this analysis sys- tem, the peak for short-chain amylose given by the RI- detector overlapped the peak for the salts in the reaction buffer. However, if glucan with a high molecular weight (>1,000 k) is produced, its peak could be easily detected. Table 1 summarizes the results. In the reaction mixtures including kidney bean BE and Bacil- lus cereus BE, no high molecular weight glucans were detected. However, glucans with Mw more than 1,000 k were detected in the mixtures including other BEs. Rhodothermus obamensis BE gave glucans with an ex- tremely high molecular weight, whereas Aquifex aeolicus BE produced glucan that was similar in size to the β-particles of NSG. A. aeolicus BE was selected for further analyses. The high-molecular-weight glucan produced by this method (Fig. 1) can be considered to be ESG, because the properties and structures of the products were similar to those of NSGs as described later. From a practical standpoint, Amylose A is prefer- able to Amylose AS-10, since the former can be easily obtained by treating starch with isoamylase (IAM), although the yield of ESG from Amylose A was lower than that from Amylose AS-10.

Eﬀect of amylomaltase

In order to solve the problem of low yield of ESG, we added amylomaltase (AM, EC 2.4.1.25) from Thermus aquaticus to the reaction mixture. The addition of AM Fig. 1. Glycogen synthesis by the IAM-BE-AM method. ↓, α-1,6- to the reaction mixture significantly improved the yield glucosidic linkage; –, α-1,4-glucosidic linkage; ◦, glucosyl residue; of ESG (Kajiura et al. 2008). The yield reached 64% ◦, glucosyl residue with reducing end; ,theα-1,4 linkage cleaved under optimal conditions. by BE; , the glucosyl residue used as an acceptor; n, number of amylose molecules initially present in the reaction mixture; m, number of synthetic glycogen molecules. Some glucosyl residues How to control the molecular weight of ESG connected by α-1,4 linkages are indicated with broken lines. (A) Short-chain amyloses are firstly prepared from starch or mal- After debranching of partially degraded starch, maltodextrin by using IAM. (B) A singly-branched molecules are produced by the action of BE on short-chain amyloses. (C) The todextrin (DE 8–9.5), by using IAM, we used it at branched molecule is preferentially used as a substrate for BE various concentrations as the substrate for BEs in the in the second reaction. (D) Branching reactions are repeated to synthesis of ESGs (Kajiura et al. 2008). At a higher produce high-molecular-weight glucans (glycogens). substrate concentration, the Mw values of ESGs were lower. By changing the concentration of substrate, we can control the Mw of ESGs. source and extraction method, and therefore they show structural heterogeneity (Table 2). The average chain Structures of ESG lengths of ESGs (A-I) tended to be slightly shorter than those of NSGs, whereas exterior chain lengths and in- We synthesized ESGs (A-I) under various reaction con- terior chain lengths were within the variations of the ditions, and compared the properties and structures of values for NSGs (Table 2). On the other hand, the the ESGs with those of NSGs (Table 2) (Kajiura et al. unit-chain distributions of ESGs and NSGs as analyzed 2008, 2010). The fine structural parameters of glyco- by high-performance anion exchange chromatography gen were the average chain length, the exterior chain after IAM treatment were slightly different from each length, and the interior chain length (Hata et al. 1984; other (Takata et al. 2009): NSGs have larger amounts Manners 1991). The structures of NSGs depend on the of long unit chains (degree of polymerization (DP): 25– 390 H. Kajiura et al.

Fig. 2. Comparison of aqueous solutions of α-glucans. (a) Waxy corn starch (0.1 g) was gelatinized in 10 mL of distilled water by heating at 100 ◦C for 30 min. The same amounts of maltodextrin (DE 8–9.5), NSG from rabbit liver and ESG-F were each dissolved in 10 mL of distilled water. (b) These solutions (1 mL) were added 10 µL of iodine reagent (52 mg of I2 and 520 mg of KI in 10 mL of water).

Table 2. Structural parameters of ESGs and NSGs.

Sample name a b c or NSG source Mw (k) CL ECL ICL

ESG A 2720 9.3 5.3 3.0 ESG B 4960 11.6 7.6 3.0 ESG C 7080 9.3 6.3 2.0 ESG D 7940 8.5 5.0 2.5 ESG E 9720 9.1 6.1 2.0 ESG F 11800 8.2 4.2 3.0 ESG G 13900 8.7 4.8 2.8 ESG H 15000 8.6 5.0 2.6 ESG I 28700 8.9 4.6 3.3

Oyster 6010 10.2 6.6 2.7 Slipper limpet 4830 9.0 5.3 2.7 Mussel 5120 10.6 6.5 3.1 Bovine liver 2030 11.9 7.0 3.9 Rabbit liver 15700 12.5 7.7 3.9 Sweet corn 19800 13.3 8.2 4.1 Fig. 3. 5% (w/v) aqueous solutions of ESGs. a Number-average chain length, CL = [Total sugars as glucose]/ [non-reducing end residues]. b Exterior chain length, ECL = CL×β-amylolysis (%)/100 + 2. c Interior chain length, ICL = CL – ECL – 1. ure 2b shows the comparison of the solutions with the addition of iodine. The solutions of amylopectin and maltodextrin turned dark blue and dark purple colors, 35) than ESGs. In other words, ESGs have narrower respectively. By contrast, the solutions of NSG from unit-chain distributions than NSGs. rabbit liver and ESG-F turned a reddish-brown color. NSG and ESG showed similar color reaction with io- Appearance of aqueous solution dine. The larger the molecular size, the deeper were Figure 2a shows a comparison of the solutions of amy- the opalescent colors of the aqueous solutions of ESGs lopectin, maltodextrin, NSG from rabbit liver and ESG- (Fig. 3). The colors of the NSG solutions also depended F. The solution of maltodextrin was clear/colorless, on their molecular sizes (data not shown). The concen- whereas the solution of amylopectin was cloudy. By con- tration of ESG also aﬀected the depth of color (Fig. 4). trast, the solutions of glycogens (NSG from rabbit liver In the concentration range of 1–10% (w/v), the higher and ESG-F) were opalescent, as described by Manners the concentration, the deeper was the color. However, (1991). All ESGs listed in Table 2 were easily soluble in in the range of 10–20% (w/v), the color of the solution water and the aqueous solutions were also opalescent, became lighter. Similar phenomena were also observed resembling NSGs (data not shown). Furthermore, Fig- for the NSG solutions (data not shown). In vitro synthesis of glycogen 391

ameters of around 80–100 nm were found. These large particles may be aggregates of smaller particles, since it is known that the β-particles sometimes associate to form larger α-particles (60–200 nm) with a rosette-like appearance (Kjoelberg et al. 1963; Manners 1991). In the image of ESG, aggregated forms (α-particles) of glycogen were not found.

Intrinsic viscosity of ESG

We determined the intrinsic viscosities of glycogens by using a capillary viscometer of Ubbelohde type. The intrinsic viscosities of ESGs and NSGs were very low (around 8 mL/g), and showed no molecular weight de- pendence (Kajiura et al. 2010). By contrast, Kitamura Fig. 4. 1, 5, 10 and 20% (w/v) aqueous solutions of ESG-F. et al. (1989) and Kitamura (1996) demonstrated that the intrinsic viscosities of amylose and amylopectin in- crease with their molecular weights. The intrinsic viscosity result suggested that ESG molecules behave as hard spheres in solution, resembling the behavior of NSG.

Digestibility of ESG

Digestibilities of maltodextrin, NSGs and ESGs were investigated according to an in vitro digestion method (Okada et al. 1990; Hashimoto et al. 2006) (Tables 3, 4). All polysaccharides were not digested by artiﬁcial gastric juice. Glycogens were digested by human salivary α-amylase, porcine pancreatic α-amylase, and rat small intestinal mucosa enzymes, the hydrolysis rates were 46–62%, 44–55%, and 60–75%, respectively. Maltodex- trin was found to be digested easier than glycogens, as expected from its lower degree of branching. The digestibility of ESG was seemed to be lowest among these polysaccharides.

Susceptibility of ESG to pullulanase and α- amylase

Pullulanase (EC 3.2.1.41) hydrolyzes α-1,6 linkages of α-1,4/1,6 glucans such as pullulan, starch, and glycogen. However, it has been shown that the enzyme only partially hydrolyzes glycogen, trimming its out- ermost branches (Kitahata 1995). Glycogen synthesized by the GP-BE method was shown to be resis- tant to pullulanase (Boyer & Preiss 1977; Kitahata Fig. 5. TEM images of ESG-C and NSG from mussel. Scale bars: & Okada 1988). Therefore, we tested the pullulanase 100 nm. susceptibilities of the ESG and NSG (Fig. 6) (Ka- jiura et al. 2008, 2010). Waxy corn starch was easily degraded into small fragments by pullulanase. How- Image of transmission electron microscopy ever, NSG from oyster and the ESG-B were barely de- (TEM) graded. This result indicates that the ESG was similar to NSG, but was unlike amylopectin. Furthermore, According to TEM images (Fig. 5), both ESG and NSG we tested the susceptibility of ESG by excess amounts have a slightly distorted circular shape, with diameters of α-amylase (Takata et al. 2009). The ﬁnal products of 20–40 nm (Kajiura et al. 2010). The shapes and sizes of α-amylase hydrolysis of NSG were glucose, maltose, were in good agreement with those of β-particles pre- maltotriose, branched oligosaccharides with DP≥4, and viously reported by Hata et al. (1983). In the image of highly branched macrodextrin molecules with molecu- NSG, a few large and irregular shaped particles with di- lar weights of up to 10 k. In the ﬁnal products of α- 392 H. Kajiura et al.

Table 3. Digestive conditions.

Reaction conditions

Substrate concentration (%) U/g-substrate pH ◦CTime(min)

Human salivary α-amylase 5 160 6.0 37 30 Artiﬁcial gastric juice (0.02M HCl-KCl) 0.7 2.0 37 100 Porcine pancreatic α-amylase 0.5 400 6.6 37 360 Rat small intestinal mucosa enzymes 0.5 43 6.6 37 180

Table 4. Digestibility of glycogen.a

Reducing sugar increased (%)

Human salivary Artiﬁcial Porcine pancreatic Rat small intestinal Sample α-amylase gastric juice α-amylase mucosa enzymes

Maltodextrin (DE 2–5) 67.2 0.0 55.5 77.7 NSG (oyster) 50.9 0.0 43.8 74.5 NSG (sweet corn) 61.8 0.0 55.3 72.2 ESG-A 46.6 0.0 43.6 60.2 ESG-C 46.0 0.0 44.2 62.9 a Reducing sugars were measured by the dinitrosalicylic acid method (Takata et al. 2009).

Fig. 7. Macrophage-stimulating activity of ESG. RAW264.7 was cultured with ESGs or lipopolysaccharide (LPS) in the presence of interferon γ. After cultivation for 48 h, NO concentration in the culture supernatants was determined.

Fig. 6. Susceptibility of α-glucan to pullulanase. ESG-B (), NSG to this role, some reports have suggested that glyco- from oyster (), and gelatinized waxy corn starch ( ) were treated gen has an immunological activity (Thorpe & Marcus with pullulanase. Mw values of hydrolyzates were analyzed by 1967; Mantovani 1981; Takaya et al. 1998). However, HPSEC-MALLS-RI. strong scientific evidence has not been obtained for the immunomodulating activity. Indeed, many scien- amylase hydrolysis of ESGs, we detected much larger tists have been annoyed by the lack of reproducibility macrodextrins (molecular weight >1,000 k). In contrast of their experimental results. This lack of reproducibil- to NSG, oligosaccharides with DP 4-7 could not be de- ity may be because their samples of glycogen have been tected in the ESG hydrolysates. These results suggest extracted from natural resources, as (1) the effect of that the α-1,6 linkages in ESG molecules are more reg- trace amounts of other materials cannot be ruled out, ularly distributed than those in NSG molecules. ESG is and (2) important characteristics of each glycogen sam- synthesized in much simpler circumstances than NSG, ple, such as the average molecular mass and the chain in which no trimming reactions occur during the syn- length, are quite different depending on the source and thetic process. This simplicity can result in a regular in- purification procedures. We have clarified the immuno- ternal structure without long spans between α-1,6 link- logical activity of glycogen by using completely pure ages. ESG with very uniform characteristics (Kakutani et al. 2007, 2008). First, we examined the immunestim- Physiological function of ESG ulating effect of ESG on NO (nitric oxide) production using RAW264.7, a murine macrophage cell line. Glycogen is well known as the energy and carbon re- The result showed glycogen enhanced NO production serves in animal cells and microorganisms. In addition of RAW264.7 (Fig. 7). The immunostimulatory activity In vitro synthesis of glycogen 393 of glycogen relates closely to its molecular weight and Boyer C.D., Simpson E.K. G. & Damewood P.A. 1982. The pos- reaches to the highest at around 5,000 k-6,500 k. Next, sible relationship of starch and phytoglycogen in sweet corn. 34: the peritoneal-exudate cells collected from C3H/HeJ II. The role of branching enzyme I. Starch/Stärke 81–85. Cori G.T. & Cori C.F. 1943. Crystalline muscle phosphorylase. mice, Toll-like receptor 4 mutants, were also activated IV. Formation of glycogen. J. Biol. Chem. 151: 57–63. by ESGs with similar profiles as RAW264.7 (Kakutani Fujii K., Takata H., Yanase M., Terada Y., Ohdan K., Takaha T., et al. 2007). Furthermore, we demonstrated by flow cy- Okada S. & Kuriki T. 2003. 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