Metabolic flux and incorporation of [2-13C]glycine into silk fibroin studied by 13C NMR in vivo and in vitro

Insect Biochem. Vol. 21, No. 7, pp. 743-748, 1991 Printed in Great Britain.All rights reserved

0020-1790/91 $3.00 + 0.00 Copyright© 1991 PergamonPresspie

METABOLIC FLUX A N D INCORPORATION OF [2-13C]GLYCINE INTO SILK FIBROIN STUDIED BY 13C NMR IN VIVO AND IN VITRO TETSUO ASAKURA,~* MAKOTO DEMURA,1 MARIKONAGASHIMA,t RYUJI SAKAGUCHI, MINORU OSANAI2 and KATSUAKIOGAWA3 tFaculty of Technology, Tokyo University of Agriculture and Technology, 2-Chome, Nakamachi, Koganei, Tokyo 184, 2Department of Biology, Kanazawa University, 1-1 Marunouchi, Kanazawa 920 and 3Research Institute for Biological Science, Katakura Industries Co. Ltd, 4-5-25 Chuo, Matsumoto 390, Japan (Received 14 February 1991; revised and accepted 23 May 1991)

Abstract--t3C NMR spectra in vivo and/n vitro of Bombyx mori silkworm larva and the posterior silk gland were observed after administration of [2-13C]glycine.In the in vivo 13CNMR spectra of larva, the administrated glycine flowed immediately from the midgut to the hemolymph and was then incorporated into the silk fibroin. This [2-t3C]glycineflux was analyzed with eight rate constants which were determined with a compartment model. The incorporation of [2J3C]glycine into the silk fibroin was also observed by t3C NMR spectroscopy by circumfusion cultivation of the posterior silk gland. The rate constant for the incorporation of glycine in vitro was determined as 7.4 x 10-4 rain -t. This was 63% of the rate constant determined in vivo. Key Word Index: silk fibroin; Bombyx mori silkworm larva; in vivo 13CNMR; [2-13C]glycinelabeling; cultivation of posterior silk gland of Bombyx mori

INTRODUCTION The biosynthetic mechanism of silk fibroin in the silkworm, Bombyx mori is unique because this fibrous protein composed mainly of glycine, alanine and serine is produced very rapidly in large quantities in the posterior silk glands (Asakura, 1987). Thus, it is very meaningful to investigate the biosynthesis of silk protein. Traditional in vitro investigations involve selective isolation of cellar components or individual enzymes. This approach has the advantage of simplicity and enables analytical and metabolic studies under highly controlled conditions. In vitro conditions, however, may not resemble the in vivo environment. Conclusions must eventually be verified by direct observation under the physiological state that characterizes the living system. Before the advent of in vivo, nuclear magnetic resonance (NMR) spectroscopy such investigations usually employed radio tracers and involved extraction and isolation of the labeled compounds of interest. N M R applications are non-invasive and allow the identification and analysis of metabolites and study of metabolic processes in vivo. More recently, excellent review of the use of N M R spectroscopy in studies of insect metabolism has been published (Thompson, 1990). By the use of both in vivo N M R and ~3C labeling techniques, we have analyzed important metabolisms with [1-~3C]glycine (Asakura et al., 1984; Asakura, 1987), [1-~3C]alanine (Asakura et al., 1984, 1985; Asakura and Murakami, 1985) [1-13C], [2-13C] or *Author for correspondence. 743

[1,2-13C]acetates (Asakura et al., 1987a) and [1-]3C]glucose (Asakura et al., 1988) in the silkworm larva related to the production of silk fibroin. Recently, the posterior silkgland isolated from 5th-instar B. mori larvae was monitored by ~3C N M R spectroscopy to determine the degree of silk fibroin production with [1-t3C]glycine and by 3tp N M R spectroscopy relative to the physiological state of the tissue in the medium (Asakura et al., 1990). It was revealed that the activity of silk fibroin synthesis for the posterior silk gland is influenced by the physiological states. However, the determination of in vivo flux or incorporation rate of the amino acids into the silk fibroin has not been analyzed. It is here that/n vivo N M R of silkworm is used to our advantage. In this paper, we will report the monitoring of [2-1aC]glycine metabolism of B. mori silkworm larva by L3CN M R in vivo and in vitro. The advantage of the use of [2-]3C]glycine is a shorter relaxation time of the 2-13C carbon which generates a greater peak intensity within limited N M R accumulation times when compared with the 1-13C carbon of [1-13C]glycine. It is possible to obtain the efficiency of in vitro production of silk fibroin in the posterior silk gland by the comparison of the rate constants between in vivo and in vitro N M R experiments. MATERIALS AND METHODS

Silkworm Bombyx mori larvae of hybrids of commercial stock,

Shuko × Rhuhaku, were reared with an artificial diet (Silk Mate, 1S and 2S, Nihon Nosan Kogyo Co., Tokyo, Japan) at 25°C in our laboratory (Asakura et al., 1990). The

744

TETSUOASAKURAet al.

4-day-old 5th instar larvae were used for measurements of in vitro and in vivo NMR spectra, respectively. In vivo N M R measurements

50 ~1 of 5% (w/v) [2-x3C]glycine (99.5 atom% 13C enrichment, Isotech Co., Miamisburg, Ohio, U.S.A.) in aqueous solution was given by oral administration to a 4-day-old 5th-instar larva after starvation for one day (Asakura et al., 1988). Immediately, the larva was placed in a sample tube of 10 mm dia, and the NMR spectra were observed at 25°C with a JEOL FX-90Q NMR spectrometer (Japan Electron Optic Laboratory, Akishima, Japan) operating at 22.5 MHz without spinning the tube (Asakura, 1987). Time interval between the oral administration and the accumulation of the ~3CNMR spectrum was within 5 min (Asakura et al., 1988). Spectral conditions are as follows (Asakura et al., 1990): 1800 or 7200 pulses, 5000 Hz spectral width, 8 K data points, 45 ° pulse angle (12.5#s) and 1 s delay between pulses. For signal locking during the acquisition, the external 7Li signal was used. Assignments of amino acid residues of the B. mori silk fibroin and metabolites were performed on the basis of the chemical shift data reported previously in detail (Asakura et al., 1984; Asakura, 1987). For assignments of the 13Clabeled metabolites, the extracts from the larva were collected as follows. 30 min after oral administration of the [2J3C]glycine, the midgut content was collected in ice-cold 80% ethanol. The suspension was centrifuged (10,000g x 10 min). The supernatant was evaporated by slowly rotating the tube under gentle vacuum, and then freeze-dried. ~3CNMR spectra of the extracts from the midgnt were observed after adjustment to pH 10, corresponding to pH in the midgut of the 5th instar larva (Eguchi et al., 1986). The hemolymph of the larva was collected in an NMR sample tube in an ice bath 30 min after injection of the [2J3C]glycine aqueous solution into the hemolymph and 13C spectra of the hemolymph were then immediately observed, maCNMR spectra of standard samples, glycine and citrate, were observed after pH adjustment with 0.1 M HC1 or NaOH.

Reclrculation pump Oxygermtor

Medium containing [2 -13 C]Gly Magnet

• Posterior sUkgland

10 mm I~ >1 NMR sample tube

Fig. I. Scheme of cultivation of circumfusion of the posterior silk gland of B. mori larvae for NMR spectroscopy. The posterior silk glands excised from four larvae were cultivated at 25°C in 8 ml of the culture medium in an NMR sample tube of 10 mm dia. Cultivation medium was saturated with an oxygen with an oxygenator through the wall of a silicone elastomer tubing. The flow rate of the medium was 2 ml/min. After pre-cultivation of the silk glands for 1 h, the culture medium was replaced by that containing 2 mg/ml of [2J3C]glycine.

in vivo NMR experimental condition and the latter fully

relaxed NMR experimental condition. The details of the correction for the individual peaks are described in the text.

In vitro N M R measurements

The integument of 4-day-old 5th-instar larva was cleaned with 70% ethanol and then, under sterile conditions, the posterior silk glands were excised and washed thoroughly with ice-cold 1.15% KC1 aqueous solution to remove adherent fat and tracheae. Kanamycin sulfate (0.05 #g/ml, Meiji Seika Co., Tokyo, Japan) was added to the culture medium (Grace's Insect Medium, Gibco Laboratory, U.S.A.) as an antibiotic. The posterior silk glands of four larvae were cultivated in 8 ml of the culture medium in an NMR sample tube of 10mm dia. as shown in Fig. 1. Oxygen was continuously and gently provided to the silk glands in vitro in the NMR tube through the wall of a silicone elastomer tubing (Asakura et aL, 1900). The flow-rate of the medium was 2 ml/min. After pre-cultivation of the silk glands for 1 h, the culture medium was replaced by that containing 2 mg/ml of [2-13C]glycine. ~3CNMR spectra were observed as a function of culture time under the same spectral conditions as mentioned above. Determination o f amounts o f t3C-labeled metabolites

The time-dependent difference '3C NMR spectra between before and after "C-labeling of B. mori silkworm was obtained. Three replicates of 13CNMR observation were done for averaging and the peak simulation assuming Lorentian was performed to resolve the over-lapped peaks (Asakura et al., 1987b). The NMR observation was performed under non-saturating conditions in order to get the spectra within limited NMR accumulation times. Thus, the amounts of ~3C-labeled metabolites were determined after the correction of the peak areas as follows. The NMR spectra of the metabolites were observed at both 1 s delay time and 50 s delay time. The former time corresponds to

RESULTS In vivo N M R

analysis o f [2J3C]glycine f l u x

The in vivo ~3C N M R spectra of a 4-day-old 5th instar larva of B. mori after oral administration of [2-13C]glycine are shown in Fig. 2. The spectra were collected in 30-120 min-blocks. Significant increases of the peak intensities after the oral administration of [2J3C]glycine occurred in the region of 40-50 ppm. The increased intensity of these peaks is due to the incorporation of the ~3C-labeled glycine into the metabolic pathway in the silkworm. Figure 3 shows the expanded t 3 C N M R spectra of Fig. 2 (40-50 ppm). A total of four peaks was observed, occurring at 46.5, 45, 43.5 and 42.5ppm for the spectra in 0-30 min block. In addition, the peak at 45 ppm moves in lower field direction and decreases its intensity with time (Fig. 3). On the contrary, the peak intensity at 43.5 ppm increases gradually. Taking into account the chemical shift values (Asakura et al., 1984), the peak at 43.5 ppm (Fig. 3) is attributed to the ~t-carbon of the glycine residue of silk fibroin. In order to clarify the assignment and initial transfer of other three peaks in the tissue, additional experiments were performed as follows. t3C N M R spectra of the extracts from the midgut were observed after adjustment of p H to 10 corresponding to p H in the midgut of the 5th instar larva (Eguchi et al., 1986). Only two peaks at 44 and 46.5 ppm attributable to glycine-C~ and methylene

Incorporation of [2-m3C]glycineinto silk fibroin

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Fig. 2. In vivo 13CNMR spectra of B. mori larva (4-day-old 5th instar) after oral administration of [2-~3C]glycine.NMR spectra were observed at 22.5 MHz without spinning the tube. Spectral conditions; 1800 or 7200 pulses, 5000 Hz spectral width, 8 K data points, 45° pulse angle (12.5#s) and 1 s delay between pulses. The assignments show amino acid residues in the silk fibroin and metabolites derived from [2-13C]glycine. carbons of citrate were observed. Moreover, these assignments were confirmed with standard samples [Fig. 3(B)]. In addition, only one peak at 42.7 ppm attributable to the ~-carbon of glycine was observed in 13C N M R spectra of the hemolymph which was collected from the larvae after [2-13C]glycine was injected into the hemolymph. This chemical shift agrees with that observed/n vivo (Fig. 3). In addition, it is well known that the methylene 13C peak of glycine shifts to low field with alkaline condition as

(A)

8~

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720-840

\

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a result of the change in the electronic distribution from the N H ~ C H 2 C O O - to the NH2CH2COOaround p K = 9.75 (Bovey, 1972), suggesting such as pH indicator. Gradual shift of the peak of 45 ppm toward lower field is presumed to be the recovery of pH in the midgut from some distortion due to the oral administration of glycine. The change of relative area of each peak with time was determined (Fig. 4), where the peak simulation assuming Lorentian was performed (Asakura et al.,

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Fig. 3. Expanded 13CNMR spectra of B. mori larva (A), and that of standard and extracted samples from the larva (B). (a) glycine at pH 1l, (b) glycine at pH 6.7, (c) extract obtained from the midgut after oral administration of [2-~3C]glycine(pH 10), (d) hemolymph collected from the larva after the treatment of [2-~3C]glycine (pH 6.7). Each spectral condition was the same as shown in Fig. 2.

746

TETSUOASAKURAet al. 1.0 <

0.5 G)

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ee

O 0

1

2

3 4 5 Time(hour)

6

O

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Fig. 4. Relative area of ]3C NMR peaks of glycine in the midgut (O), in the hemolymph (©) and the silk fibroin (11) and citrate in the midgut (I--l)as a function of observation time. Relative area of each peak was determined by the peak simulation assuming Lorentzian and corrected on the basis of ]3C NMR relaxation times as mentioned in Materials and Methods. Solid lines were calculated with the optimum constants listed in Table 1. 1987b). The correction of the peaks of the ~3C-labeled metabolites was performed by taking into account the N M R relaxation times (see Materials and Methods section). The peak intensity of the glycine Cot carbon observed at pH 6.7 was increased by 1.50 times after correction, glycine C~ at pH 6.7 ~ 1.50. Similarly, glycine Cot at pH 10 ~ 1.88, glycine C~ of the residues of the silk f i b r o i n ~ 1.0, and citrate - C H 2 - at pH 10 ~ 1.42. The amounts of the glycine in the midgut rapidly decreases and that of citrate is low under these conditions. It is clear that the rapid transport of glycine from the midgut to the hemolymph is followed by incorporation into the silk fibroin. In vitro N M R analysis of incorporation of [2-z3C]glycine into silk fibroin Figure 5 depicts the time-dependence of z3C N M R spectra (a--c) of the culture medium including the posterior silk glands after addition of [2J3C]glycine in the medium. The peak intensity of [2J3C]glycinelabeled silk fibroin increased with time, in contrast with the decrease of the [2J3C]glycine peak. Also, the peak due to the Cot carbon of the serine residue of silk fibroin slightly increased. However, the rate of incorporation of glycine into the silk fibroin in the posterior silk gland is obviously faster than that of interconversion between glycine and serine in the silk gland. The relative areas of the Cot peaks of [2-13C]glycinesilk fibroin and [2-13C]glycine in the culture medium were plotted against cultivation time (Fig. 6), where the N M R relaxation factors of glycine Cots at pH 6.7 and that incorporated in the silk fibroin were 1.50 and 1.0, respectively. Incorporated glycine increased symmetrically to the decrease of free glycine.

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Fig. 5. The time-dependence of in vitro 13CNMR spectra of the silk glands cultured in the medium containing [2J3C]glycine. Cultivation time; (c) 0-2 h, (b) 6-8 h and (a) 12-14h. Spectrum (d) shows the pre-incubation of silk glands without [2-t3C]glycine.Spectral conditions were the same as in Fig. 1. The assignments of glycine, serine and alanine are correspondence to the silk fibroin. [Fig. 7(B)], it is possible to discuss the kinetics of in vivo glycine transport with the data of Fig. 6. Here, the relative area of each 13C N M R peak is considered to be proportional to the amount of each component from [2J3C]glycine. As shown in Fig. 7(B), the relative amounts of the 13C-labeled components in the four compartments are denoted as X], X2, X3 and X4, where subscripts, 1-4, represent glycine in the midgut, citrate in the midgut, glycine in the hemolymph, and glycine incorporated in the silk fibroin, respectively. The rate constant of each step is denoted as Kv (v = 1-8). The kinetic equations with time, t, are written as follows: dX1/dt = - ( K 1 + Kz + Ks)XI + K3X2 + KsX3 (1) dX2/dt = K2Xl -- (K3 + K4)Xz (2) 1.0 ~ , ~

_m

DISCUSSION On the basis of the time-dependent peak intensities shown in Fig. 4, a model of transport of glycine in B. mori larva is proposed as shown in Fig. 7(A). In the midgut, a large portion of glycine is transported through the midgut membrane into the hemolymph, while a smaller portion is converted to citrate. The glycine in the hemolymph is absorbed into the posterior silk gland where it is incorporated into the silk fibroin. According to the compartment model

o o

2. 4

6 8 lO 1 2 1 4 16 Timelhour)

Fig. 6. In vitro incorporation of [2-13C]glycineinto the silk fibroin in the posterior silk glands as a function of cultivation time. Relative area of [2-t3C]glycinein the silk fibroin (O) and the medium (0) was determined by the peak simulation. Relative area of each peak was corrected in the same manner described in the legend of Fig. 4. Solid lines were calculated one.

747

Incorporation of [2-13C]glycine into silk fibroin

(A)

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/

(B) Oral aCknkllstratiOlt [ of glyclne ~

IK 8

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Midgut

[

Posterior Silk

gland

^1

J K3

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[Glyxoaine] I

"

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Fig. 7. Model of [2J3C]glycine transport through the midgnt into the silk fibroin (A) and four-compartment model for the glycine transport (B). Closed circles of C in the structural formula means 13C-labeled carbons. X and K are amount of ~3C-labeled metabolites and in vivo rate constant for various transport of glycine or citrate (see the text). d X 3 / d t = K I X I - (K5 + K6 + KT)X3

(3)

d X 4/dt -- K6X 3

(4)

These can be solved in X with eight parameters, K, computationally by the Runge-Kutta-Gill method (Conte and de Boor, 1980). In this case, curve-fitting by a simplex method (Nelder and Mead, 1965) for the data in Fig. 4 was performed in accordance with equations (1)-(4) in order to obtain the optimum parameters. The rate constants obtained are listed in Table 1. As shown in Fig. 4, the solid lines calculated with the parameters listed in Table 1 agrees well with Table 1. Rate constants* of [2-13C]glycine transport in the B. mori larva Rate constant x 10-2/min-t K1 K2 K3

K, K5 K7

7.20 5.62 10.37

0.62 3.17

0.12 0.06

K s 0.43 *Rate constants were defined as equations (1-4) described in the text. The relative area of each t3CNMR peak was determined after the correction mentioned in Materials and Methods.

the change of intensities. The value of K~/Ks is much larger than that of K2/K3, indicating that the rate of absorption through the midgut membrane into the bemolymph is faster than the conversion from glycine to citrate in the midgut. Although K6, which represents the incorporation of glycine in the bemolymph into the fibroin is a relatively small value, another flux of glycine in the hemolymph, KT, is much lower than K6. This suggests that the efficiency of incorporation of glycine into the silk fibroin is high in this stage of the larva. Asakura et al. (1988) have reported the presence of glycine ( ~ ) serine conversion in B. mori larvae with [1-13C]glucose by in vivo 13C NMR. The peak due to the C~ carbon of the serine residue of silk fibroin slightly increased in vitro (Fig. 5), indicating there is still a system of glycine (*-~) serine conversion in the posterior silk gland itself. However, the incorporation of glycine into silk fibroin is considered to be faster than the rate of the interconversion in the silk gland. By assuming that the incorporation of glycine into silk fibroin in vitro follows frst order kinetics as in equation (4), the rate constant was determined by the curve-fitting as 7.4 x 10 - 4 min -~. This value was 63% of the corresponding value (K6) which was determined in vivo N M R of the silkworm larvae (Table 1). Shimura (1978) and Shigematsu and Koyasako (1961) have also demonstrated the importance of glycine concentration or the combination of amino acids for in vitro biosynthesis of the silk fibroin with ~4C-glycine. However, the rate of silk fibroin synthesis has not been determined under non-destructive conditions. In this study, continuous N M R observation of the tissue, the silk gland or whole larva, enabled determination of the kinetics with [2-13C]glycine.

REFERENCES

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TETSUOASAKURAet al.

Asakura T., Tateno A., Kawaguchi Y., Hamano K. and Mukaiyama F. (1987a) Biosynthesis of Bombyx mori silk fibroin from [1-13C], [2-t3C] and [1,2-t3C] sodium acetate. J. Seric Sci. Jpn 56, 38-44 (in Japanese). Bovey F. A. (1972) In High Resolution NMR of Macromolecules, 254 pp. Academic Press, New York. Chinzei Y. (1975) Induction of histolysis of ecdysterone in vitro: breakdown of anterior silk gland in silkworm, Bombyx mori. Appl. ent. Zool. 10, 136-138. Conte S. D. and de Boor C. (1980) In Elementary Numerical Analysis. McGraw-Hill. Eguchi M., Kuriyama K. and Daimon H. (1986) High alkalinity and function of proteases of digestive juice from

the silkworm, Bombyx mori. J. Seric. Sci. Jpn 55, 46-53 (in Japanese). Nelder J. A. and Mead R. (1965) A simplex method for function minimization Computer J. 7, 308-313. Shigematsu H. and Koyasako T. (1961) Silk fibroin synthesis in Bombyx mori posterior silk gland/n vitro. Bull. Seric exp. Stn Jpn 17, 295-317 (in Japanese). Shimura K. (1978) Synthesis of silk proteins, In The Silkworm: An Important Laboratory Tool (Edited by Tazima Y.), pp. 189-211. Kodansha, Japan. Thompson S. N. (1990) NMR spectroscopy: its basis, biological application and use in studies of insect metabolism. Insect Biochem. 20, 223-237.