Calcium influences the stability and conformation of rotavirus SAH glycoprotein VP7 expressed in Dictyostelium discoideum

Journal of Biotechnology 50 (1996) 149-159

Calcium influences the stability and conformation of rotavirus SAl 1 glycoprotein VP7 expressed in Dictyostelium discoideum Kerry R. Emslie”, M. Barrie Coukella,b, Debra Birch”, Keith L. Williams”,* “MUCAB

(Macquarie

University Centre for Analytical Biotechnology), School of Biological Sciences, Macquarie University, Sydney, N.S. W. 2109, Australia bDepartment of Biology, York University, 4700 Keele St., North York, Ont. M3J IP3, Canada

Received 18 December 1995; revised 24 May 1996; accepted 3 June 1996

Abstract We have previously reported expression of the rotavirus outer capsid glycoprotein, VP7, in the relatively new expression host, Dictyostelium discoideum. To optimise yields of recombinant VP7, we examined the role of CaZf since stability of both VP7 and mature rotavirus during a rotavirus infection are calcium-dependent. Low micromolar levels of free extracellular Ca*+ were required to maximise yields of VP7 in D. discoideum whilst levels of VP7 were reduced following depletion of intracellular Ca* + reserves using A23187 and EGTA. Immunoblot analysis suggested that VP7 was being degraded in an intracellular compartment. Immunoprecipitation with a conformation-dependent neutralising antibody confirmed that EGTA-induced Ca’ + chelation alters the conformation of VP7. These results suggest that stability of VP7 is dependent on maintaining adequate levels of intracellular Ca*+ and that conformational changes in VP7 which occur following depletion of Ca2 + reserves induce rapid proteolysis of the protein. Since these results establish conditions for expressing optimal levels of VP7 in the correct conformation they have important implications for the development of a subunit vaccine based on recombinant VP7. Keywords:

Ca 2+ , Rotavirus VP7; Dictyostelium;

Conformation;

Protein expression

1. Introduction

* Corresponding author. Tel: + 61 2 8508212; fax: + 61 2 8508174; e-mail: [email protected]

Rotavirus is the major cause of viral gastroenteritis in humans and animals. The mature rotavirus virion is composed of a core, an inner capsid and an outer capsid comprising the two

0168-1656/96/$15.000 1996 Elsevier Science B.V. All rights reserved PZI SOl68-1656(96)01557-X

K.R.

150 Table 1 VP’J-specific

Type

D35

Rabbit clonal Rabbit clonal Mouse clonal

60

159

(‘I ~1. Joumui

of Biorechnology

50 (1996)

149--159

antibodies

Antibody

A28

Emslie

Source

Specificity poly-

SAI I rotavirus

poly-

Denatured

mono-

Calcium-independent; dependent on a VP7 conformation requiring disulphide bonds; does not recognise a secreted form of VP7 (M, -37.5 x IO’ under reducing conditions) which is expressed in D. discoideum (Emslie et al., 1995a); non-neutralising Serotype 3-specific; calcium-dependent: conformation-dependent; neutralising

Mouse monoclonal

proteins

VP7: detects

all forms of VP7

major neutralizing antigens, VP4 and VP7. Both VP4 and VP7 are possible candidates for use in a subunit vaccine against rotavirus. During a rotavirus infection, single-shelled rotavirus particles assemble in a dense viroplasm within the cytoplasm and then bud through the endoplasmic reticulum (ER), where the outer capsid protein VP7 is located. Final assembly of the outer capsid of the mature virus takes place within the ER (Bellamy and Both, 1990). Both assembly and stability of the mature virus particle are calcium-dependent. During a rotavirus infection, the high levels of Ca2+ in the ER (Somlyo et al., 1985; Baumann et al., 1991) favour assembly of the outer capsid whilst this process is inhibited in Ca2 + -deprived rotavirus-infected cells (Shahrabadi and Lee, 1986). In addition, calcium chelation results in loss of the outer shell of rotavirus (Cohen et al., 1979). The stability of the outer capsid protein, VP7, is also calcium-dependent as increased degradation of this glycoprotein has been observed in Ca2 + -deprived rotavirus-infected cells (Shahrabadi and Lee, 1986; Shahrabadi et al., 1987; Poruchynsky et al., 1991). It is believed that Ca2 + is required for VP7 to acquire the correct protein conformation prior to final virus maturation in the ER. Whilst VP7 lacks a classic Ca2 + -binding domain, its conformation is clearly influenced by Ca2+ as neither viral VP7 nor herpes simplex virus-l-expressed VP7 are recognised by neutralising monoclonal antibodies following Ca2 + chelation (Dormitzer and Greenberg, 1992).

A.R. Bellamy, Auckland, New Zealand A. R. Bellamy, Auckland, New Zealand Shaw et al., 1986; Svensson et al., 1994

Shaw et al., 1986

We have used the simple eukaryote Dictyostelium discoideum to express the rotavirus glycoprotein, VP7 (Emslie et al., 1995a). This relatively new expression system is an attractive and economic alternative for producing large quantities of antigen for use in a subunit vaccine (Emslie et al., 1995b). Because Ca’ + plays such a significant role in the stability of rotavirus VP7, here we examine the importance of Ca2+ in optimising yields of recombinant VP7 and provide evidence that the conformation of recombinant VP7 is influenced by Ca2 +

2. Materials and methods 2. I. Materials Chemicals and antibody conjugates were obtained from the following sources: ethyleneglycol-bis-(P-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA), 2[N-morpholinolethanesulphonic acid (MES), leupeptin, 1,4-diazabicyclo[2.2.2]octane (DABCO) (Sigma Chemical Company); calcium chloride (Merck); AEBSF.HCl (Calbiochem); N-glycosidase F, A23 187 (Boehringer Mannheim); Triton X-100 (BDH); peroxidaseconjugated sheep anti-rabbit and anti-mouse immunoglobulin (Silenus Laboratories). Suppliers of other materials used are detailed in Emslie et al. (1995a). Antibodies specific for VP7 are detailed in Table 1.

K.R. Emslie et al. /Journal

of Biotechnology 50 (1996) 149-159

2.2. Buffers The following buffers were used. For cell culture, Buffer A (20 mM potassium phosphate, pH 6.5); Buffer B (20 mM MES-NaOH, pH 6.5); Buffer C (20 mM MES-NaOH, 1 mM EGTA, pH 6.5). For cell lysis and biochemical analysis, sodium dodecyl sulphate (SDS) lysing buffer (1.5% (w/v) Tris, 7.2% (w/v) glycine, 0.5% (w/v) SDS, 1 PM E-64, pH 8.6); TBS-TX-E (25 mM Tris, 150 mM NaCl, 1 mM MgSO,, 1% (v/v) Triton X-100, 1 PM E-64, 10 mM NaN,, pH 7.4); TBS-Ca-TX-E (TBS-TX-E containing 3 mM CaCl,).

151

ml aliquots) and each adjusted to give final additional concentrations of 0.0, 0.3, 0.6, 1.3 and 2.7 mM CaCl,. The suspensions were shaken at 150 r-pm at 21 + 1°C for 6 h. At zero hour and regular time intervals after, l-ml portions of cell suspension were transferred to microcentrifuge tubes. The cells were pelleted (1300 x g, 4 min). The supernatant was further centrifuged (15 000 x g, 4 min) to remove any cellular debris, and then transferred to clean tubes containing the cysteine proteinase inhibitor, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64; final concentration, 1 PM). Cell pellets were solubilised in SDS lysing buffer (1 ml) and stored with supernatant samples at - 2O’C.

2.3. Strains The transformed cell line, HU2767, which is derived from the axenic D. discoideum NP2 fucosylation mutant, HU2860, has been described previously (Emslie et al., 1995a). To prepare spores of HU2767, amoebae were grown at 21°C with Micrococcus luteus (Wilczynska and Fisher, 1994) on SMj5 agar (Dittrich et al., 1994) containing 10 pg of active geneticin per ml, and permitted to fruit. Spore caps were picked into sterile salt solution (Bonner, 1947) and washed ( - 1000 x g, 5 min). Spores were then resuspended in axenic growth medium (Watts and Ashworth, 1970) supplemented with 5% (v/v) dimethyl sulphoxide (DMSO) and frozen at - 80°C in aliquots of - 5 x lo6 or - 5 x lo7 spores. Klebsiella aerogenes cells were grown (21°C 3 days) on SM agar (Sussman, 1966) harvested with a glass spreader, suspended in 10% PBS, pH 7.2 and 15% glycerol, aliquoted and frozen at - 20°C for up to 3 months. 2.4. Conditions for growth on solid medium and starvation in phosphate buffer HU2767 spores (5 x 106-5 x lo7 ) were inoculated together with K. aerogenes on 1 1 of SM agar. After 3-4 days growth, cells and remaining bacteria were harvested and resuspended in 200 ml of Buffer A (final concentration l-2 x lo7 cells per ml) (Emslie et al., 1995a). The cell suspension was dispensed into five 125-ml conical flasks (40-

2.5. Conditions for growth in liquid culture and starvation in MES or MESiEGTA buffer Thawed K. aerogenes cells were diluted in Buffer B or Buffer C to give an OD,,, reading of - 1.3 when diluted a further lo-fold. Thawed HU2767 spores were added (final concentration - 2 x lo5 viable spores per ml), and the culture shaken (150 rpm, 21°C). The spores germinate in 3-4 h and then the amoebae grow exponentially with a doubling time of - 3 h to a density of -2 x lo7 ml-‘. For VP7 expression, amoebae were grown to a density of 3 -8 x lo6 cells per ml, harvested by centrifugation (200 x g, 4 min), washed three times by centrifugation in Buffer B or Buffer C, and resuspended in the same buffer (final concentration 1.1 x lo7 cells per ml). Exactly 0.9 ml of cell suspension was placed in wells of a 24-well plate (Costar) and then supplemented with CaCl, in Buffer B to give the desired free Ca2+ concentration in a total volume of 1.0 ml. When present, the final concentration of EGTA was 0.9 mM. Free Ca2+ concentrations were determined by a computer programme based on the calculations of Fabiato and Fabiato (1979). The plate was shaken ( - 135 rpm) at room temperature on a Ratek Orbital Mixer. At the times indicated, 150-~1 portions of cell suspension were transferred to microcentrifuge tubes (taking care to first wash clumped cells from the sides of the wells). The cells were pelleted (1300 x g, 2 min or in some experiments,

152

K.R. Emslie et 01. Journd

c!f Biotrhoiog~

15 000 x g, 10 s) and aliquots (120 ~1) of each supernatant were transferred to clean tubes containing E-64 (final concentration, 10 PM). Cell pellets were washed in Buffer B (1 .O ml), aspirated dry, solubilized in TBS-Ca-TX-E (30 jrl) and stored with supernatant fractions at - 20°C. In Ca2+ depletion experiments, A23 187 was added to the cell suspensions from a freshly prepared 500 PM stock in DMSO. This concentration of DMSO alone ( w 0.2%) had no effect on total VP7 levels.

50 (1996) 149-1.59

temperature and then washed three times with TBS-Ca-TX-E. Cells (3 x 10’) were lysed in 100 irl of TBS-Ca-TX-E and then incubated for 2 h with the mAb 159 conjugated to the Protein A beads. The beads were washed twice with TBSCa-TX-E, three times with TBS-TX-E, and VP7 was eluted with TBS-TX-E containing 10 mM EGTA. The beads were then boiled in Laemmli sample buffer for 3 min.

3. Results 2.6. N-Glycosidase F digestion, electrophoresis and Western immunoblot anal_vsis For N-Glycosidase F digestion, 5 x 10’ cells were lysed in TBS-Ca-TX-E and then incubated (2 h, 37°C) in 50 ~1 of TBS-TX-E containing 8 mM EDTA, 0.5 mM CaCl,, with and without 0.5 units of N-glycosidase F. For SDS-polyactylamide gel electrophoresis (PAGE), cell lysates and culture supernatants were diluted with Laemmli buffer (Laemmli, 1970) with or without 5% (v/v) mercaptoethanol for separation of proteins under reducing or non-reducing conditions, respectively. Samples were boiled for 3 min prior to loading on a 10% gel. For Western Blot analysis, electrophoresed proteins were transferred to nitrocellulose as described previously (Emslie et al., 1995a) and VP7 was detected using either the polyclonal antibody, A28 (1: lOOO), or the mAb 60 (1:20) followed by peroxidase-conjugated sheep anti-rabbit or antimouse immunoglobulin (1: lOOO), respectively. The public domain NIH Image software package available through Internet was used to estimate yields of VP7 by comparing densitometric scans of Western Blots containing recombinant VP7 and standard SAl I virus samples. The total cell protein of lysates was estimated using a Bio-Rad Protein Assay Kit. 2.7. Immunoprecipitation oj’ recombinant with neutralising mAb 159

VP 7

Pre-swollen Protein A Sepharose CL-4B beads were incubated with culture supernatant containing mAb 159 in TBS-Ca-TX-E for 2 h at room

3.1. iTfji?ctof’ Ca’ + on expression of’ VP7 in cells gro\ivl on solid medium and starved in phosphate bl@ We have developed a simple method for expressing recombinant rotavirus SAll VP7 in D. discoideum. Expression is controlled by the actin promoter, which is switched on during starvation. Briefly, the transformed cells are grown together with K. aerogenes on a peptonebased agar, harvested and then shaken in a phosphate buffer (Buffer A) containing 2.7 mM CaCl, (Emslie et al., 1995a). Ca*+ was included in the phosphate buffer since it affects VP7 stability in other systems. Using these growth and starvation conditions, three forms of recombinant rotavirus SAI 1 VP7 were detected ranging in relative molecular mass (M,) under reducing conditions from 35.5 x lo3 to 37.5 x 103. The smaller forms were intracellular whilst the largest form was secreted. In a subsequent experiment, Ca*+ was omitted from the starvation buffer since we reasoned that the insolubility of calcium in the phosphate buffer would render it largely unavailable to the cells. Surprisingly, in the absence of added Ca2 + , the yield of VP7 was lower and secreted VP7 was not detected. To study this effect in more detail, cells were grown as before, harvested in Buffer A and then aliquots adjusted to give final CaCl, concentrations ranging from 0 to 2.7 mM. When the cells were initially resuspended in Buffer A (0 h), VP7 was detected as a single intracellular band under reducing conditions of M, 35.5 x lo3 (Fig. 1). In the absence of added calcium, this band decreased

K.R. Emslie et al. 1Journal of Biotechnology 50 (1996) 149-159

in intensity over the subsequent 6 h and VP7 was not detected in the medium. On addition of 0.34 mM CaCl,, some VP7 was secreted during the subsequent 6 h whilst at higher Ca2+ concentrations, the yield of VP7 increased predominantly due to a further increase in secreted VP7 and significant production of an intracellular form of M, 37 x lo3 under reducing conditions (Fig. 1). Whilst VP7 was consistently secreted in the presence of Ca2+, the relative levels of intracellular and secreted VP7 varied. This lack of uniformity between experiments may be due to heterogeneity in the D. discoideum cell population as cells harvested under these growth conditions inevitably cover a wide range of developmental stages. To examine, more precisely, the influence of Ca*+ on VP7 expression in D. discoideum, the growth and starvation conditions were changed as follows. First, the cells were grown as a homogeneous population by shaking them with K. aerogenes in a simple buffer. Second, expression of VP7 was synchronised by harvesting the cells in the mid-exponential phase of growth, washing them free of bacteria and resuspending them in a starvation buffer. Using these conditions, cell growth and starvation are clearly delineated, and

IP

CaCI,

(mbl) Time (hours)

SIMIP 0 0

SILP 0 6

SI,P S,tP S,,P 0.34 0.67 1.36 6

6

6

s,

2.7 6

Fig. I. Effect of calcium on the production of different forms of recombinant VP7 by HU2767 cells starved in phosphate buffer. Amoebae were grown with K. aerogenes on SM agar, harvested, resuspended in Buffer A, supplemented with O-2.7 mM CaCI,, and shaken as described in Materials and methods. Since Ca2 + is relatively insoluble in phosphate buffer, the concentration of free Ca2+ will be lower than the total calcium concentrations shown. After 0 and 6 h of incubation, samples were processed to obtain pellet (P) and supematant (S) fractions. Reduced proteins were resolved by SDS-PAGE and VP7 was detected by immunoblot analysis using polyclonal Ab A28. M, ( x 10W3) of the protein standards in lane M are indicated on the left. Similar results were obtained in two other experiments.

153

expression of VP7 during the growth phase is minimal. Finally, MES or MES/EGTA buffers were used so that free Ca* + concentrations could be adjusted accurately. 3.2. Low micromolar levels of free extracellular Ca”

significantly increase yields of VP7

In a preliminary experiment, cells were grown with bacteria in Buffer B, washed and resuspended in Buffer B containing EGTA and free Ca2 + concentrations ranging from < 1 nM to 4.0 mM. Samples were taken at regular intervals up to 21 h and the yield of VP7 and relative levels of VP7 in the cell lysate and the supernatant estimated by serial dot and Western blot assays using mAb 60 and polyclonal A28 Levels of VP7 were again influenced by Ca’+; however, the effects differed in several respects to those observed in the phosphate buffer. Most notably, low micromolar concentrations of free Ca*+ were sufficient to improve the yield of VP7 since VP7 levels were similar at free Ca*+ concentrations from 0.01 to 4.0 mM (data not shown). Small amounts of the largest A4, secreted form were detected after incubating cells for more than 4 h in the presence of at least 0.1 mM free Ca2 + . However, in contrast to the Ca2 + -dependent secretion observed following starvation in phosphate buffer, the amount of secreted VP7 did not increase with increasing free Ca2 + concentrations. Furthermore, secretion of VP7 was not observed consistently and was restricted to experiments where cells were harvested late in the growth phase ( > 8 x lo6 cells per ml). Again, this may reflect an effect on harvesting the cells at a different developmental stage. However, since the secreted form of VP7 does not contain neutralising epitopes (Emslie et al., 1995a), these studies were focused on the intracellular form of VP7. To ensure optimal Ca*+ concentrations for VP7 expression, in subsequent studies the level of free Ca2 + in the starvation buffer was adjusted to 0.1 mM. To examine the effect on VP7 yields of partially depleting cells of Ca2 + , the following experiment was conducted. Cells were grown with bacteria in Buffer B or Buffer C, washed in the same buffer, and incubated for 22 h in Buffer C and either < 1

154

K.R.

Emslie

et ul.

Jourwd

, P s,, P s,, P s,, P s,, P s,, P s,

01

24822 Time (hours)

34 -28

B

C

-34

-34

-28

-28

_

,PL!s 0

+

4

Time (hours) Fig. 2. Effect of extracellular free Ca’+ levels during growth and starvation on the yield of recombinant VP7. (A, B) HU2767 cells were grown in a shaken suspension of K. arrogenes in (A) Buffer B (-EGTA) or (B) Buffer C (+ EGTA) washed free of bacteria in the same buffer, resuspended in Buffer C, and dispensed into two aliquots. One suspension received no additional calcium (upper panel) while the second suspension was adjusted to an extracellular free Ca” ’ concentration of 0. I mM (lower panel). The suspensions were shaken and, at the times indicated, samples were processed to obtain pellet (P) and supernatant (S) fractions. (C) A lysate 01 amoebae starved for 4 h in the presence of Ca’ + was incubated without ( - ) or with ( + ) N-Glycosidase F as described in Materials and methods. Non-reduced proteins were fractionated by SDS-PAGE and VP7 was detected by immunoblot analysis using mAb 60. Protein standards (M, x IO - ‘) are indicated on the right. Similar results were obtained in another experiment.

nM or 0.1 mM free Ca2 + (Fig. 2A and Fig. 2B). In both cases, two major intracellular forms of VP7 were observed. This is in contrast to starvation in phosphate buffer (Fig. l), when two intracellular forms of VP7 were observed only in the presence of at least 1.35 mM CaCl,. Under reducing conditions these two forms had apparent M, values of 35 x lo3 and 37 x 10” (data not shown).

of Biotechnology

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As reported previously (Emslie et al., 1995a), under non-reducing conditions, these two forms migrated more quickly with apparent M, values of 32.5 x lo3 and 35.5 x lo”, respectively. N-Glycosidase F digestion confirmed that the larger M, form, which has the same apparent M, as SAll viral VP7 (Emslie et al., 1995a), is the N-glycosylated derivative of the smaller A4, form (Fig. 2C). In the absence of EGTA during growth, a low level of VP7 was expressed by the cells (Fig. 2A, 0 11). When these cells were starved in the presence of 0.1 mM free Ca”~, the yield of both the glycosylated and non-glycosylated forms of intracellular VP7 increased up to 4 h and then stayed relatively constant up to 8 h (Fig. 2A, lower panel). In contrast, when the starvation buffer contained < 1 nM free extracellular Ca*+, the yield of VP7 stayed relatively constant up to 4 h and then decreased slightly in the 4-8 h period (Fig. 2A, upper panel). In both cases, there was significant cell lysis after 22 h and although full length N-glycosylated VP7 was not detected, smaller mAb 60-positive polypeptides of M, 28 x 1O3 and 32 x lo3 were present in the pellet and supernatant of cells exposed to 0.1 mM Ca2+ these were probably degradation products. In comparison, the level of VP7 in cells grown in the presence of 1 mM EGTA was lower than the level in cells grown in the absence of EGTA (compare Fig. 2A and Fig. 2B, 0 h). After starving these cells in 0.1 mM free Ca2 + , the yield of both the glycosylated and non-glycosylated forms of intracellular VP7 increased significantly over the first 4 h (Fig. 2B, lower panel). Again, very little VP7 was detected in the medium except some smaller M, forms at 22 h following significant cell lysis (data not shown). When shaken in buffer containing < 1 nM free Ca2+, the yield of VP7 increased slightly over the first 4 h (Fig. 2B, upper panel) but no VP7 was detected after 22 h (data not shown). In the experiment shown, some supernatant samples contained full length N-glycosylated VP7; however, this was not reproducible. These results confirmed that higher yields of recombinant VP7 were obtained when the cells were maintained in low micromolar levels of free extracellular Ca2 +

K.R. Emslie et al. J Journal of Biotechnology 50 (1996) 149-159

3.3. Depletion of intracellular Ca2+ stores results in a rapid loss of expressed VP7

To determine if Ca”+ was required to maintain the stability of VP7 after expression, we examined the effect of Ca2+ depletion on levels of VP7. Cells were grown with K. aerogenes in Buffer B, washed and then starved for 4 h in 0.1 mM Ca2 + . In three independent experiments, the VP7 yield at this time was estimated as 513 + 55 ng per 10’ cells or 0.2% of the total cell protein. The cells were treated with A23187 and EGTA or Buffer B and then shaken for a further 4 h. During the first 4 h of starvation, there was a significant increase in the yield of the two major forms of VP7 as expected, and in control cells, intracellular VP7 levels stayed relatively constant from 4 to 8 h (Fig. 3A). Several forms of VP7 were also detected at a low level in the supernatant during this time period. In contrast, yields of VP7 in cells treated with ionophore and EGTA decreased substantially (Fig. 3B). VP7 was not detected in the

I P 0

SII

P

4

SIIP

SIIP

5

SllP

6

SllP

7

SI

a

Time (hours) Fig. 3. Effect of Ca *+ depletion on the yield of recombinant VP7. HU2767 cells were grown in a shaken suspension of K. aerogenes in Buffer B, washed free of bacteria, and resuspended in Buffer B. The cell suspension was adjusted to 0.1 mh4 CaCIz, dispensed into two ahquots, and shaken. At the times indicated, samples were processed to obtain pellet (P) and supematant (S) fractions. Immediately prior to the 4 h timepoint, EGTA and A23187 (final concentrations: 1 mM and I PM, respectively) were added to one suspension (B) and an equal volume of Buffer B was added to the other suspension (A). SDS-PAGE, immunoblot analysis and protein standards as in Fig. 2. Similar results were obtained in two experiments using cells grown on solid medium and starved in Buffer A.

155

supernatants of these samples nor was it detected when a cocktail of protease inhibitors (100 PM AEBSF, 10 pg ml-i leupeptin, 1 PM E-64, final concentrations) was included in the starvation buffer (data not shown). Whilst Ca2+ depletion clearly resulted in a significant loss of VP7 over the subsequent 4 h, even at the first time point after addition of ionophore and EGTA, there was a difference in the level of VP7 (compare the 4-h timepoints in Fig. 3A and Fig. 3B). Because harvesting and washing the cells prior to lysis took 10-l 5 min, it was possible that Ca’+ depletion was affecting VP7 stability during this period. Based on this observation, the experiment was repeated but samples were collected just prior to addition of A23187 and EGTA, and then after 5, 15, 30, 60 min and 4 h. The harvesting and washing protocol was also modified so that the entire procedure from sampling to cell lysis took < 4 min. In this experiment, the level of VP7 in control cells increased slightly from 0 to 60 min and further still over the next 3 h (Fig. 4A). In contrast, the VP7 level in cells exposed to ionophore and EGTA dropped dramatically within 5 min and stayed low for the first 15 min. As in control cells, the level then increased slightly to 60 min, but was finally depleted during the subsequent 3 h. In two independent experiments, 60 min after ionophore treatment, the intensity of the N-glycosylated VP7 band was 31-42% of that from control cells. Whilst small amounts of VP7 were detected by mAb 60 in the supernatant of control cells, VP7 was not detected in the supernatant within 5 min of exposing treated cells to ionophore and EGTA nor was it detected at later times (Fig. 4B). To determine if the decrease in detectable VP7 was due to loss of recognition by mAb 60 (which only detects VP7 with intact disulphide bridges and does not detect the larger M, secreted form (Emslie et al., 1995a)), we also examined the cell lysates and culture supernatants with polyclonal antibody, A28, which, although less sensitive than mAb 60, detects all forms of VP7 (Table 1). The pattern of VP7 detection with A28 was very similar to that with mAb 60 both in the pellet and supernatant fractions (Fig. 4C and Fig. 4D). Thus the rapid decrease in detectable VP7 following depletion of intracellular Ca2 +

K.R. Emslie et rrl. : Journul qf Biocerhnology 50 (1996) 149- 159

I56

-34

B

-28

.34

D

-20

the same study, recognition by the neutralising mAb 159 was shown to be independent of added Ca'+ However, since the ELISA was not done in an EGTA buffer, it is possible that residual micromolar levels of Ca*+ were sufficient to retain the conformation of VP7 in a form which is recognised by mAb 159. Because results from the present study suggested that Ca*+ is required to maintain the stability of expressed VP7, we examined the effect of Ca*+ depletion on recognition of recombinant VP7 by the neutralising mAb 159. VP7 was immunoprecipitated with mAb 159 in the presence of 3 mM CaZ+ (Fig. 5). Both the N-glycosylated and non-glycosylated forms of recombinant VP7 were recognised by mAb 159 and remained bound to mAb 159 during washes with

l-.-.JII-+II-+II-+II-+I~

0

5

15 30 Time (minutes)

60

240

Fig. 4. Depletion of intracellular Ca’+ leads to rapid degradation of expressed VP7. HU2767 cells were grown in a shaken suspension of K. aerogenes in Buffer B, washed free of bacteria, and resuspended in Buffer B. The cell suspension was adjusted to 0.1 mM CaQ, dispensed mto two aliquots, and shaken at room temperature for 4 h. At zero time, the two suspensions were sampled, then one suspension ( + ) was treated with EGTA and A23187 (final concentrations: I mM and 1 FM, respectively) while the second suspension (~ ) received an equal volume of Buffer B. Additional samples were taken at the times indicated. Samples were processed to obtain pellet (A,C) and supernatant (B,D) fractions. Proteins were fractionated by SDS-PAGE under non-reducing (A,B) or reducing (CD) conditions and VP7 detected by immunoblot using mAb 60 (A,B) or polyclonal Ab A28 (CD), respectively. Protein standards as in Fig. 2. Similar results were obtained in three additional experiments.

be explained by increased secretion of VP7 or by an alteration in the VP7 molecule leading to loss of recognition by mAb 60.

cannot

3.4. Ca” affects the conformation recombinant and viral VP7

of

Using an enzyme-linked immunosorbent assay (ELISA), we have shown previously that recognition of Dictyostelium-expressed VP7 by the conformation-dependent neutralising mAbs 4C3 and 4F8 is Ca’ + -dependent (Emslie et al., 1995a). In

28 123456 Fig. 5. Effect of EGTA on binding of recombinant VP7 to the conformation-dependent neutralising mAb,159. Amoebae (3 x 106) starved for 4 h in the presence of Ca2 + were lysed in TBS-Ca-TX-E. Pre-swollen Protein A Sepharose CL4B beads were incubated with culture supernatant containing mAb 159 in TBS-Ca-TX-E for 2 h and then washed with the same buffer. The cell lysate was then incubated for 2 h with the mAb 159 bound to the Protein A beads. The beads were pelleted and then washed sequentially with TBS-Ca-TX-E. TBS-TX-E, TBS-TX-E-EGTA and then boiled in Laemmli buffer. Lane I: cell lysate. Lane 2: cell lysate after incubation with mAb 159 bound to Protein A beads. Lane 3-5: supernatant from beads washed with TBS-Ca-TX-E, TBS-TX-E, TBSTX-E-EGTA, respectively. Lane 6: supernatant after boiling beads in Laemmli buffer. VP7 was eluted from the beads in the presence of 10 mM EGTA (lane 5). MAb 159 remained bound to the beads in the presence of EGTA (lane 5) and was eluted (arrow) by boiling the beads in Laemmli buffer (lane 6). SDS-PAGE, immunoblot analysis and protein standards as in Fig. 2. Similar results were obtained in three additional experiments.

K.R. Emslie el al. 1 Journal of Biotechnology 50 (1996) 149-159

TBS-Ca-TX-E and TBS-TX-E. However, upon addition of 10 mM EGTA, VP7, but not mAb 159, was efficiently eluted from the Protein A beads. MAb 159 was only eluted from the Protein A beads after boiling in Laemmli buffer (Fig. 5). This result indicates that binding of VP7 to the neutralising mAb 159 is a Ca* + -dependent process supporting the concept that Ca*+ is affecting the conformation of VP7.

4. Discussion We have described previously a simple method for producing milligram quantities of the rotavirus outer capsid glycoprotein, VP7, in D. discoideum (Emslie et al., 1995a). The current study has demonstrated that low micromolar levels of free extracellular Ca2 + during expression enhance the yield of VP7. In addition, depletion of cellular Ca* + reserves results in a rapid loss of VP7 as detected by either the mAb 60, which only recognises intracellular VP7 containing intact disulphide bridges (Svensson et al., 1994), or the polyclonal Ab A28, which also recognises both denatured VP7 and a larger h4, secreted form of VP7 (Emslie et al., 1995a). Although enhanced secretion of some proteins has been reported following exposure of mammalian cells to A23187 (Suzuki et al., 1991), in the case of VP7, a secreted form was not detected by polyclonal Ab A28 (Fig. 4C and Fig. 4D) even when the protease inhibitors, AEBSF, E-64 and leupeptin were incorporated into the starvation medium. If VP7 was transported out of the ER and through the Golgi before degradation, the M, of VP7 should increase to that of the secreted form (Emslie et al., 1995a). However, treatment with A23187 and EGTA did not change the M* of undegraded VP7 (Figs. 3 and 4). These results suggest that, following calcium depletion, VP7 is either degraded within the ER or a post-ER pre-Golgi compartment, or is transported out of the ER and rapidly degraded in another compartment or in the starvation buffer before the higher A4, form of VP7 is detected. Although an ER-located degradation pathway has not been characterised in D. discoideum, such a proteolytic pathway was impli-

151

cated in the rapid degradation of two endogenous intracellular D. discoideum glycoproteins, cyclic nucleotide phosphodiesterase and its inhibitor following depletion of cellular Ca2+ reserves (Coukell and Cameron, 1990; Coukell et al., 1992) and is now well established in response to Ca2 + depletion in mammalian systems (Wileman et al., 1991). Why should depletion of intracellular Ca2+ lead to rapid intracellular degradation of VP7? The ER is the major intracellular reservoir of Ca*+ ions in non-muscle cells (Baumann et al., 1991) and is involved in the regulation of cytoplasmic Ca 2+ levels. Although a direct measurement of the ER Ca*+ concentration is difficult to obtain, it has been estimated as > 5.0 mM in mammalian liver parenchymal cells (Somlyo et al., 1985). Since VP7 is synthesised and then retained in the ER during a viral infection, it is normally exposed to the high Ca* + levels of the ER. Whilst there is no direct evidence that Ca2+ binds to VP7, Ca*+ is required to stabilise the rotavirus outer capsid which contains VP7 and VP4 (Cohen et al., 1979) and is also necessary to preserve conformation-dependent neutralisation epitopes on VP7 following expression in several eukaryotic hosts including D. discoideum (Fig. 5; Dormitzer and Greenberg, 1992; Emslie et al., 1995a). Attempts to determine the intracellular location of D. discoideum-expressed VP7 were complicated since VP7 had a dramatic effect on the morphology of the ER. In contrast to control cells, the ER in transformants was largely depleted of ribosomes (unpublished observations). Immunofluorescence and immunoelectron microscopy studies localised recombinant VP7 to a tubular network in a reticular pattern throughout the cell which was almost certainly the ER (unpublished observations). However, in the absence of a D. discoideum ER marker, it was not possible to conclusively establish that this network was the ER. Nevertheless, since treatment with A23187 and EGTA will rapidly deplete ER and cellular Ca*+ levels, this is likely to influence the conformation of VP7, perhaps making it more susceptible to resident proteases. Residual Ca2+ levels may be sufficient to maintain some VP7 molecules in the correct conformation and this could ac-

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count for the observation that Ca2 + depletion did not lead to complete loss of VP7. This report documents the requirement for Ca2+ to optimise yields of an intracellular recombinant glycoprotein, rotavirus VP7 which is expressed at high level in D. discoideum. The results also suggest that the high levels of Ca2+ within the ER play an important role in maintaining recombinant VP7 in a conformation which is recognised by neutralising antibodies. Whilst secretion of recombinant proteins may be favoured as it simplifies subsequent purification procedures, it may be advantageous to retain proteins, such as VP7, in the intracellular compartment in which they are normally found and this option should be considered when expressing recombinant intracellular proteins for biotechnological applications.

50 (19%)

14% 159

Coukell, M.B. and Cameron, A.M. (1990). Calcium depletion of Diotyostelium cells selectively inhibits cyclic nucleotide phosphodiesterase synthesis at a post-transcriptional step. J. Cell Sci. 97. 6499657. Coukell, M.B.. Cameron, A.M. and Adames, N.R. (1992). Involvement of intracellular calcium in protein secretion in Dic~~mteliunz discoideum. J. Cell Sci. 103, 371 --380. Dittrich, W., Williams. K.L. and Slade, M.B. (1994). Production and secretion of recombinant proteins in Dictyostelium dkoideum. Bio!Technology 12, 614.-617. Dormitzer, P.R. and Greenberg, tion induces a conformational pes simplex virus-l -expressed

H.B. (1992). Calcium chelachange in recombinant herrotavirus VP7. Virology 189,

Acknowledgements

X28 832. Emslie. K.R., Miller, J.M., Slade. M.B., Dormitzer, P.R.. Greenberg, H.B. and Williams, K.L. (1995a). Expression of the rotavirus SAI I protein VP7 in the simple eukaryote Dictwstelium discoideum. J. Viral. 69, 1747- 1754. Emslie, K.R., Slade, M.B., and Williams, K.L. (1995b). From virus to vaccine: developments using the simple eukaryote, Dictyostelium di.voideum. Trends Microbial. 3, 4766479. Fabiato, A. and Fabiato, F. (1979). Calculator programs for computing the composition of the solution containing multiple metals and ligands used for experiments in skinned

We thank Drs. Gerry Both, Dick Bellamy and Harry Greenberg for providing the SAl 1 rotavirus and VP7 gene, the polyclonal antisera A28 and D3.5, and the mAbs 60 and 159, respectively. We also thank Jenny Minard and Anne Cameron for technical assistance. This research was supported by a grant to K.R. Emslie and K.L. Williams from the Australian National Health and Medical Research Council and a grant to M.B. Coukell from the Natural Sciences and Engineering Research Council of Canada (NSERC).

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