Journal of Biotechnology, 23 (1992)55-70

55

© 1992ElsevierSciencePublishers B.V. All rights reserved0168-1656/92/$05.00

BIOTEC 00714

Processing of the prepropeptide portions of the Bacillus amyloliquefaciens neutral protease fused to Bacillus subtilis a-amylase and human growth hormone during secretion in Bacillus subtilis Akira Nakayama, Hiroaki Shimada *, Yoshio F u r u t a n i and Masaru H o n j o Biotechnology Laboratory, Life Science Institute, Mitsui Toatsu Chemicals, Inc., Togo, Mobara, Japan

(Received 15 July 1991;revisionaccepted3 September1991)

Summary A set of nested 3'-terminal deletions of the prepropeptide of the Bacillus amyloliquefaciens neutral protease gene was constructed. Alpha-amylase and human growth hormone were secreted using these truncated genes in Bacillus subtilis. The level of the secreted a-amylase varied with the region for the truncated prepropeptide contained in the fusion gene but was independent of its length. Even though length of the prepropeptide varied, the mobilities of secreted a-amylases were the same as that of the control a-amylase derived from the a-amylase clone, pTUB4 (Yamazaki et al., 1983). Analyses of the secreted Nterminal amino acid sequences confirmed that they were all identical to that of the authentic one. Precursor proteins of the a-amylase were found in the cell-associated fraction, suggesting that the prepropeptide portion was processed during secretion. On the other hand, the N-terminus of hGH secreted using one of these prepropeptide portions varied by 1 to 4 additional N-terminal amino acid residues derived from the junction sequence between the sequence for propeptide portion and mature hGH or from C-terminal region of the propeptide portion. These results suggest that the prepropeptide portion can be generally processed even in the heterogeneous fusion. A probable mechanism of processing and maturation of the fusion gene products is also discussed. Correspondence to: A. Nakayama, BiotechnologyLaboratory, Life Science Institute, Mitsui Toatsu Chemicals, Inc., 1144,Togo, Mobara 297 Japan. * Present address: Mitsui Plant BiotechnologyResearch Institute, Sengen2-1-6, Tsukuba, 305 Japan.

56 Neutral protease gene; Prepropeptide; Processing; Human growth hormone; aAmylase; Bacillus subtilis

Introduction

Bacillus amyloliquefaciens secretes large amounts of alkaline protease and neutral protease into the culture medium. These genes are well investigated and have been shown to have the prepropeptide coding region (Vasantha et al., 1984; Shimada et al., 1985; Wang and Doi, 1986). The corresponding sequence in the neutral protease gene has been shown to encode 221 amino acid residues preceding the mature enzyme. This prepropeptide region is suggested to be composed of a prepeptide (27 amino acid residues) which functions as a secretion signal, and a propeptide of 194 amino acid residues. Though it has been shown that the propeptide plays an important role in the formation of a higher structure to produce enzymatically active subtilisin (Ikemura et al., 1987), the precise function of the propeptides of both proteases is still unclear. We have previously reported secretion of a-amylase by the entire prepropeptide region of the B. amylofiquefaciens neutral protease gene. In this case, efficient secretion and exact maturation of a-amylase were observed (Honjo et al., 1986). This result showed that B. subtilis cells can accurately cleave the propeptide even in a chimeric protein. Processing of the propeptide, however, does not seem to be essential to protein transport in such a case, suggesting that deletion of the entire propeptide fused to the a-amylase was not intrinsic for secretion and might be done by the enzymatic activity involved in the processing of the authentic neutral protease precursor. If so, C-terminally truncated propeptides fused to heterogeneous proteins might not be deleted after cleaving the prepeptide during secretion. In view of this, we constructed a series of fusion genes each of which was composed of a 3'-terminally-deleted propeptide region and the gene for mature a-amylase or human growth hormone (hGH) to examine the processing of the precursor during secretion. In this paper, we describe secretion of B. subtilis a-amylase and hGH using C-terminally truncated prepropeptides from B. amyloliquefaciens neutral protease in B. subtilis. We also discuss the probable manner of the processing of the truncated propeptides preceding a-amylase and hGH during secretion.

Materials and Methods

Bacterial strains, plasmids and medium Strains and plasmids used in this work are listed in Table 1. Two X Luria broth was used as liquid medium. Composition of this medium is as follows: 2.0% bacto-tryptone (Difco), 1.0% yeast extract, 0.2% glucose and 1.0% NaCI. One

57 TABLE 1 Bacterial strains and plasmids Strains/plasmid B. subtilis 207-25 B. subtilis N325 B. subtilis MT430 pNP150 pTUB4 pPA33

Remarks r-, m-, arnyE, aro1907, lys21, leuA8, recE4 r-, m-, amyE, npr, apr ~°w,aro1907, lys21, leuA8 r-, m-, amyE, npr, apr I°w, aro1907, lys21, leuA8, degQ(BamF)

Source/reference Yamane, K. Honjoet al. (1985)

B. amyloliquefaciens npr clone B. subtilis amyE clone Coding hybrid protein composedof entire prepropeptide of B. amyloliquefaciens NPR and B. subtilis mature a-AMY

Honjo et al. (1984) Yamazaki et al. (1983)

Honjo et al. (1987)

Honjo et al. (1985)

a NPR, neutral protease; a-AMY, a-amylase; npr, neutral protease gene; amyE, a-amylase gene. percent of soluble starch (Wako Pure Chemicals, Osaka, Japan) and 1.5% agar were supplemented to this medium when used as starch plates. Construction o f fusion plasmids containing a truncated prepropeptide with the aamylase gene

A set of nested deletions of the propeptide-coding region was generated by Ba131 nuclease digestion after cleaving pNP150 with PvuI. The fragment encoding the mature a-amylase of B. subtilis was prepared from pTUB4 and subcloned into pUC12. This sequence in pAM29, the resultant hybrid plasmid, was isolated by digestion with SstI and H i n d l I I (group 1) or with S m a I and P v u l I (group 2). The resultant 1.9 kb fragment was ligated into Bal31-treated pNP150 after making it blunt-ended using T4 D N A polymerase (Fig. 1A). Construction o f fusion plasmids for secretion o f h G H

The region encoding the mature a-amylase in pNPA84, which is one of the hybrid plasmids containing a region for the entire prepeptide and the propeptide portion composed of 21 amino acid residues preceding a-amylase gene, was replaced by the h G H gene according to two procedures as follows: (1) The plasmid pNPA84 was treated with B a m H I and Klenow fragment to make it blunt-ended. A mature h G H gene prepared from pGH20 (Honjo et al., 1986) was ligated into the B a m H I site which had been blunted with Klenow fragment. The plasmid obtained, was designated phGH928 (Fig. 2). (2) The plasmid pNPA84 was sequentially digested with B a m H I , mung bean nuclease and HindlII. T h e resulting fragment was ligated with a chemically synthesized double stranded DNA, 5'-CCTTCCCAACTATACCACTITCGCGCCTATTCGATACGCAAGTCTACGTGCTCACC G A C T A C A T C A G C T G A A G C T - 3 ' , encoding the sequence for the first 22 amino acid residues of the mature hGH. The resultant plasmid containing a fusion gene encoding a chimeric protein that is composed of the C-terminally truncated

58

Alul ~ o R I

( pTUB4 ~ n._mmylflsegene

(A) •

. promoter

pUC13

/ / I

N ' 'oz

\.

~:~neulral I~1 protease ~ gene

pNP 150

~-::"

,.o_,b/

pt~i.ot=

\\. Rm

IEooR, /

~

~

m

(\

~ (B)

~1~

'J"

~ ~

,,.

F-'---~SstI I-Smal ~ L'BarnH!

p..,?,.. ^.

\\~;'-"

promoter'S"

......

/ ~

/

EcoRI

IA,o,.EooR, '~

\, Ao

Pvul BAL31

M

"."-~

Nsma; jZ~HindEI

I HI omyla e

SstI Sinai BamHl

5stI, Hind/[[ T4-DNApolymerase or (Groupl) Sinai ( partial ) Pvu/[ (Groupll)

r-mature (x-amylase

- - - -- GAGCT~CGC C C~GGG~GAT CC C C IC_T T AC AGC A --1 2 3 Fig. 1. Construction of the amylase-secretion plasmid and its relevant structure. (A): Schematic representation of plasmid construction. Entire and deleted prepropeptide region of the neutral protease is indicated by an open box and the region for the mature a-amylase is shown by a cross-hatched sequence. (B): D N A sequence and deduced amino acid sequence in the junction region of pAM29. The N-terminal amino acid of the mature a-amylase is shown as amino acid number 1.

prepropeptide (entire prepeptide and first 21 amino acid residues of propeptide) and hGH was designated phGHlll0. The remainder of the mature hGH gene from pGH20 was inserted into the PvulI site of phGHlll0. The resultant plasmid was designated phGH84 (Fig. 3).

Transformation of B. subtilis cells Plasmids were introduced into B. subtilis cells according to the procedure described by Chang and Cohen (1979).

59

BamH I

0ro0 o:,#/ lr

EcoRI

.... ,,.e f

pNPA84

~

/

~

~-~--Hind IH n~r,t~a/se

"'u''

{

pGH20

~J--EcoRV

~ / ~ B a m H

,

/

--BamH I

[

_ Klenowfragment

~.~ BarnHI

EcoRI

- hGH I.. hGH

I Ligation BamHI

prepro'reg~~ promo,t~.~

/

Klenow

fragment

I

I

ph:H~;8~

BE~RHV

1~=-amylase

(B) fMet Gly • GTG.GGT . . . . . . . .

Gin Gly I]e Asn Set Met Phe Pro Thr CAA.GGG ATC AAT TCT ATG. TTC CCA ACT -- - - .

prepro-region

hGH

Fig. 2. Construction of the plasmid phGH928 and its relevant structure. (A): Schematic representation of plasmid phGH928 construction. The genes in the figure are indicated as follows: (El), the promoter region of the neutral protease gene; ( I ) , head portion of the neutral protease gene; (13), mature a-amylase gene; (11), junction region; ([]), mature hGH gene. (B): DNA sequence and deduced amino acid sequence of the junction region contained in phGH928.

SDS-PAGE and Western blot analysis SDS-polyacrylamide gel electrophoresis (PAGE) was carried out according to Laemmli (1970). Western blot analysis was performed using the IMMUN-BLOT T M assay kit (BioRad Lab.). Rabbit antiserum raised against B. subtilis a-amylase was prepared in our laboratory.

6O

(A)

Ba ., prepro-r^eg~#

~

-amylase

~l~ngUotlaIse

Hind Ill

BamH I

I

Mung bean nuclease

Hind Ill

Ligation prepro-region ~,~synthesized DNA ~ H i n d Ill Y , , / J - ~ . ~ . neutral prornoter~/~ " Pvui i r ~ protease II ~

v

u

EcoRI

vu,,

/

gAP

~

....

~

pGHZU ~ E c o R

pr°m°terL//~"

V

BarnH I

I Pvu II, EcoRV

I Ligation prepro'reg~n~

~

I hGH~

~

II/EcoRV

(B) fMet Gly Gin Gly Phe Pro Thr .GTG GGT ............ CAA,GGG,TTC CCA ACT .............

i

~prepro-region

hGH Jjunction~

Fig. 3. Construction of the plasmid p h G H 8 4 and its relevant structure. (A): Schematic representation of phGH84 construction. Symbols are as follows: ( B ) , the promoter region of the neutral protease gene; ([]), head portion of the neutral protease gene; ([]), mature a-amylase gene; ( • ) , junction region; ([]), mature h G H gene. (B): D N A sequence and deduced amino acid sequence of the junction region of phGH84.

61 TABLE 2 Length of remaining prepropeptide sequence in each fusion gene and secreted a-amylase activity Plasmid

Group

Length of remaining prepropeptide region (bp) a

Secreted amylase activity (U ml-1) b

pNPA105 pNPA73 pNPA71 pNPA107 pNPA102 pNPA74 pNPA84 pNPA58 pNPA86 pNPA152 pNPA133 pNPA155 pNPA85 pPA33

1 1 1 1 1 1 2 2 2 2 2 2 2

130 230 250 260 300 350 150 170 190 240 240 270 340 660

3.5 0.5 0.1 0.6 2.5 5.5 7.1 6.0 6.8 5.3 1.7 0.1 2.8 4.8

Constructed plasmids were introduced into B. subtilis 207-25. Amylase-producing colonies were screened on starch plates. Hybrid plasmids isolated from the transformants which produce various levels of a-amylase activities are listed. They were classified into Group 1 and Group 2 according to the sequence of junction between the region for the truncated prepropeptide and a-amylase (group 1, -Arg-Pro-Gly-Ile-Pro-;group 2, -Gly-Ile-Pro-). a The length of the prepropeptide coding region was determined by Southern blot analysis. Sau3AI fragments from the plasmids were hybridized with the AvaI-PvuI fragment of pNP150 (shown in Fig. 1A). b Amylase activity in the culture medium was measured after each transformant was cultivated for 10 h at 37 ° C in 2 × Luria broth.

Southern blot analysis P l a s m i d s u s e d S o u t h e r n blot analysis w e r e p r e p a r e d as d e s c r i b e d in t h e footn o t e o f T a b l e 2. T h e s e p l a s m i d s w e r e d i g e s t e d with Sau3AI c o m p l e t e l y a n d t h e r e s u l t a n t d i g e s t e d mixtures w e r e s u b j e c t e d to a g a r o s e gel e l e c t r o p h o r e s i s . Southe r n blot analysis was c a r r i e d o u t a c c o r d i n g to t h e m e t h o d o f S o u t h e r n (1975) a f t e r t r a n s f e r r i n g o n t o a n i t r o c e l l u l o s e m e m b r a n e . T h e f r a g m e n t c o r r e s p o n d i n g to t h e p r e p r o p e p t i d e r e g i o n in p N P 1 5 0 was u s e d as a p r o b e .

Detection and assay o f activities A m y l a s e p r o d u c i n g colonies on starch p l a t e s w e r e d e t e c t e d by f o r m a t i o n o f halos using 0.16 m M K I - 0 . 0 1 6 m M 12 solution. T h e activity o f a - a m y l a s e was assayed a c c o r d i n g to B e r n f e l d (1955). T h e r e d u c i n g sugars l i b e r a t e d f r o m starch by a - a m y l a s e w e r e m e a s u r e d by r e d u c t i o n o f 3,5-dinitrosalicylic acid. O n e unit of a m y l a s e activity is t h e a m o u n t of activity which l i b e r a t e s 1 m m o l o f m a l t o s e p e r rain at 37°C. T h e c o n c e n t r a t i o n o f h G H was m e a s u r e d by t h e s o l i d - p h a s e e n z y m e i m m u n o a s say ( E I A ) m e t h o d d e s c r i b e d previously ( H o n j o et al., 1986).

62

Protein purification The secreted amylase was sequentially purified by ammonium sulfate precipitation, DEAE-Sepharose (Pharmacia) and Bio-gel P100 (BioRad Lab.) column chromatography. Secreted hGH from B. subtilis was purified by DEAE-cellulose (Whatman DE-52) column chromatography, CM-cellulose (Whatman CM-52) column chromatography and separation on SDS-PAGE. Proteins were recovered from SDS-PAGE by the procedure described previously (Nakayama et al., 1987).

Amino acid sequence analysis Amino acid sequences from the N-terminal of the secreted a-amylase and hGH were determined by the Edman-degradation method using the model 470A sequencer (Applied BioSystems).

Results

Secretion of a-amylase using truncated prepropeptide We constructed a set of nested deletions of B. amyloliquefaciens neutral protease gene as described in Materials and Methods to confirm whether C-terminally deleted propeptide could be processed during secretion of heterogeneous fusion protein or not. These truncated prepropeptide regions were ligated to the mature a-amylase gene to construct chimeric genes (Fig. 1A). The resulting fusion plasmids were introduced into B. subtilis 207-25, an a-amylase deficient strain. Transformants were obtained which secreted a-amylase activity into the culture medium. Levels of secreted a-amylase varied according to the plasmids harbored in the transformants. Each plasmid was isolated and the size of the truncated prepropeptide region contained in it was estimated by Southern blot analysis. As shown in Table 2, a series of fusion plasmids varying in size were obtained. The level of a-amylase activity varied for each plasmid. No linear correlation was, however, found between sizes of the truncated prepropeptides and secreted a-amylase activity levels (Table 2). No differences in growth rate among transformants were observed (data not shown). We used two groups of hybrid plasmids, group 1 and group 2, which contain different junctions between the truncated prepropeptide and the a-amylase region. The sequence, -Arg-Pro-Gly-Ile-Pro-, was encoded by the junction in the plasmids of group 1 and the sequence, -Gly-Ile-Pro-, in those of group 2, respectively (Fig. 1B). Although, efficiency of secretion might be affected by differences in the junction between the prepropeptide region and a-amylase gene, diversity on secretion level of a-amylase was found in both groups (Table 2). The plasmids pNPA74 and pNPA84 permitted high-level accumulation of the a-amylase activity, whereas the plasmids pNPA105 and pNPA133 did not. We chose these plasmids as two types of plasmids responsible for hyper- and hypo-pro-

63 1

27 ~,

pNPA74

MGLGK . . . . . PGVQA AENPQLKENLTNFVPKHSLVQSELPSVSDKAIKQYLKQN 107 GKVLKGNPSERLKLIDQTI'DDLGYKHPRYVPVVNGVPVKDS~ ITA 1 27 ~ 42

pNPA105

MGLGK. . . . . PGVQA AENPQLKENLTNFVPr~"~ ITA

1

27 ,.

48

pNPA84

MGLGK. . . . . PGVQA AENPQLKENLTNFVPKHSLV [ ]

pNPA133

MGLGK. . . . . PGVQA AENPQLKENLTNFVPKHSLVQSELPSVSDKAIKQYLKQN

1

ITA

27 ,~ 82

KAIKQYLKQNGKVLKGNPSERLKLID ~

ITA

Fig. 4. Amino acid sequences encoded in the truncated prepropeptides contained in plasmids, pNPA74, pNPA105, pNPA84 and pNPA133. Junction sequences and N-terminal portion of mature a-amylase are boxed and underlined, respectively. Arrows indicate the cleavage site of the prepeptide.

duction of the a-amylase activity and analyzed by DNA sequencing. As shown in Fig. 4, pNPA74, pNPA105, pNPA84 and pNPA133 contained the portion of the prepropeptide encoding 107, 42, 48 and 83 amino acid residues, respectively. Size and N-terminal amino acid sequence of the secreted a-amylase The a-amylases secreted by several transformants were purified. Their molecular size were compared with each other and with the control a-amylase produced by B. subtilis harboring pTUB4 using SDS-PAGE. As shown in Fig. 5, a-amylases from all transformants tested showed the same mobility, suggesting that each prepropeptide portion could be deleted during secretion. The size of a-amylase derived from pTUB4 is smaller than that of authentic B. subtilis a-amylase because the 3'-terminal region for the enzyme is deleted in pTUB4 (Yamazaki et al., 1983). On SDS-PAGE of the purified a-amylase, a larger protein (approx. 66 kDa) was observed (Fig. 5). This protein was considered to be mutated inactive a-amylase derived from the host strain because the host strain harboring pTUB4 also secreted this 66 kDa protein. Western analysis using antiserum vs B. subtilis a-amylase also suggested this (data not shown). Secreted a-amylases from pNPA84, pNPA133 and pNPA74 were analyzed for their N-terminal amino acid sequences. The result indicated that they were all identical to that of the authentic a-amylase, Leu-Thr-Ala-, which has been reported by Miints~il~i and Zalkin (1979). Detection of a-amylase precursor in the cell-associated fraction The transformants harboring fusion plasmids, pNPA84, pNPA74 and pPA33, were grown and lysed with lysozyme. The resultant cleared lysates were analyzed

64

1234567

Fig. 5. SDS-PAGE of the purified cu-amylase. Cells of B. subtilis N325 harboring pTUB4, pNPA58, pNPA84, pNPA86, pNPA155, and pPA33 were grown to stationary phase. Secreted cY-amylase from each culture was purified as described in the text. Lane I: size markers (bovine serum albumin, 66 kDa; egg albumin, 45 kDa; and trypsinogen, 24 kDa). Lane 2-6: purified cu-amylases derived from pNPA58 (lane 2), pNPA84 (lane 31, pNPA86 (lane 41, pNPAl55 (lane 5), pPA33 (lane 6), and pTUB4 (lane 7). The plasmid pTUB4 is a clone of the B. subtilis a-amylase gene.

Fig. 6. Immuno-detection of the precursor cu-amylase in the cell-associated fraction. Cells of B. subtilis N325 harboring pTUB4, pNPA84, pNPA74 and pPA33 were grown to stationary phase and harvested by centrifugation. Cleared cell lysates of these cells were subjected to SDS-PAGE. Western analysis using a-amylase antiserum was performed as described in the text. Lane 1, B. subtilis N325 host control; lane 2, N325(pTUB4); lane 3, N325(pNPA84); lane 4, N325tpNPA74); and lane 5, N325cpPA33).

65 by Western blotting using antiserum vs B. subtilis a-amylase. The result (Fig. 6) indicated that there were various sizes of positive bands larger than the mature a-amylase. The mobility of each band coincided with the size of each prepropeptide-a-amylase fusion suggesting that the precursor fusion-proteins were present in the cell and were processed during secretion.

Secretion of hGH by the prepropeptide portion in B. subtilis B. subtilis 207-25 carrying plasmid pNPA84 yielded the highest level of a-amylase secretion in this study. This plasmid contains an artificial junction between the region for the N-terminal 21 amino acid residues of the propeptide and the mature a-amylase gene. However, as described above, the precursor of a-amylase was exactly processed and authentic mature a-amylase was secreted into the culture medium. Ohmura et al. (1983) have reported that the authentic B. subtilis a-amylase precursor is synthesized as a preproenzyme. Since the a-amylase gene used in this work was derived from B. subtilis, a specific machinery for the maturation of the authentic a-amylase might function even in such fusion gene products. Thus, we examined the secretion of hGH of which gene was fused to the prepropeptide portion contained in pNPA84. For this purpose, we constructed a plasmid phGH928 as shown in Materials and Methods and Fig. 2A. The sequence of precursor hGH encoded by the fusion gene contained in phGH928 is also shown in Fig. 2B. Bacillus subtilis strain MT430 harboring phGH928 could secrete hGH-antigen into the culture medium efficiently. The details on the secretion of the hGH-antigen by this plasmid have been described elsewhere (Nakayama et al., 1987). We purified the hGH-antigen and determined the N-terminal amino acid sequence. The result revealed that the secreted hGH-antigen was a mixture of N-terminally extended hGHs. The additional sequences were Ile-Asn-Ser-Met- (50%), Asn-Ser-Met- (25%) and Met-hGH (25%) (Fig. 7), which were derived from the junction sequence between the propeptide portion and the mature hGH gene. This suggested that B. subtilis could process the propeptide portion even in the secretion of the heterologous protein. In this case, it might be possible that the amino acid sequence derived from the junction served as the sequence that could function as the cleavage site by chance. If so, it could be considered that removal of the junction region coding -Ile-Asn-Ser-Met from the plasmid phGH928 might cause secretion of the authentic hGH or the hGH variant with the propeptide portion. To confirm this, we constructed such a plasmid, designated phGH84. The plasmid phGH84 contained a sequence, GGG coding Gly, as a junction between the prepropeptide region and the mature hGH sequence (Fig. 3B). This plasmid was introduced into the B. subtilis strain MT430 and the secretion of hGH was analyzed. Transformant cells harboring phGH84 permitted efficient secretion of hGH-antigen (78 mg 1-1) into the culture medium which was comparable to those carrying phGH928 (80 mg 1-1). Secreted hGH-antigen from B. subtilis MT430 (phGH84) was also purified and its N-terminal amino acid sequence was determined. The result revealed that the secreted hGH was a mixture of hGH with N-terminal extensions similar to the case

66

(A) plasmid

precursor a.a. sequences

..........................................................................

1

phGH928

MGLGK

27

1

phGH84

48

-- VQAAEN

LVQ~'~'~

27

FPT---

48

MGLGK-- VQAAEN--LVQr~FPT---

(B) plasmid

N-terminal sequences %raito( mol% )

..........................................................................

phGH928

~

F P T--FPT--~ ] FPT---

50 % 25% 25%

..........................................................................

phGH84

LVQ [ ] VQ [ ] []

F P T--F PT--FPT---

25% 25% 50%

...........................................................................

Fig. 7. Precursor amino acid sequences deduced from DNA sequences and N-terminal extensions of secreted hGH. Cells of B. subtilis MT430 harboring phGH928 and phGH84 were cultured at 30 o C for 16 h. Secreted hGH was purified by the method described in the text. Purified hGH was subjected to analysis of the N-terminal amino acid sequence. Boxed and underlined sequences are the junction and N-terminal of authentic hGH, respectively. Percent ratio indicates molar fraction of the indicated variant to the total secreted hGH variant. o f p h G H 9 2 8 . T h e s e a d d i t i o n a l s e q u e n c e s w e r e Gly- (50%), V a l - G l n - G l y - ( 2 5 % ) a n d L e u - V a l - G l n - G l y - (25%), in which a d d i t i o n a l p e p t i d e s w e r e d e r i v e d f r o m t h e j u n c t i o n (Gly) a n d C - t e r m i n a l p o r t i o n o f t h e p r o p e p t i d e ( L e u - V a l - G l n - ) p r e c e d i n g t h e m a t u r e h G H (Fig. 7), suggesting t h a t the p r o p e p t i d e p o r t i o n c o u l d b e d e l e t e d d u r i n g s e c r e t i o n i n d e p e n d e n t l y o f the j u n c t i o n s e q u e n c e .

Discussion T r u n c a t e d p r e p r o p e p t i d e of t h e B. amyloliquefaciens n e u t r a l p r o t e a s e c o u l d allow s e c r e t i o n o f B. subtilis a - a m y l a s e a n d h G H in B. subtilis. W h e n a - a m y l a s e was s e c r e t e d using t h e t r u n c a t e d p r e p r o p e p t i d e , t h e p r e c u r s o r was p r e c i s e l y p r o c e s s e d a n d t h e a u t h e n t i c m a t u r e e n z y m e was f o u n d in the c u l t u r e m e d i u m (Fig. 5). Since p r o t e i n s which w e r e c o n s i d e r e d to b e p r e c u r s o r s as well as m a t u r e a - a m y l a s e w e r e d e t e c t e d in t h e c e l l - a s s o c i a t e d fraction (Fig. 6), t h e p r e c u r s o r

67

4 3

phGH928

>

(B)

< o a

I

;>.

-2

""

mS

i SLVQG

NSMF

-4

50

100

RESIDUE RUMEER

4 3 >-

phGH84

2

0

(S)

.

.

.

.

•

.

o

rr" a

-I

~'-

m2

3::

-3

P

SLVQGF

-4

5'0

100

RESIDUE NUMBER

Fig. 8. Hydropathic profile of the precursor hGH. Hydrophobicity analysis of the region preceding authentic mature hGH was performed according to the method of Rose and Roy (1980). The symbols in the figure are as follows: (A), cleavage site of prepeptide; (B), cleavage site of propeptide. Bold arrow indicates a cleavage site producing the dominant N-terminal extension.

seemed to accumulate in the cell, probably in the membrane fraction, and was accurately processed during secretion. On the other hand, the truncated prepropeptide yielded N-terminally extended hGHs. These N-terminal extensions were derived from the sequence preceding the mature hGH region such as the junction or C-terminal of the propeptide portion. This suggests that the fusion protein, composed of propeptide portion and hGH could be recognized and deleted by processing machinery during secretion in B. subtilis. The analyses of secreted hGH variants derived from phGH928 and phGH84 suggested that the processing was not dependent upon the specific amino acid sequence. The hydropathy profiles of the precursor hGH encoded by both plasmids are shown in Fig. 8. The cleavage site of the propeptide portion might have some relationship to the hydrophobicity since the cleaved sites for dominant species were present in the most hydrophobic region preceding mature hGH in both cases. Diversity of the N-terminal extensions of the secreted hGH suggested that the cleaving enzyme involved in the processing of the propeptide portion was

68 rather ambiguous in its recognition of the cleavage site, or that an exopeptidase deleted N-terminal remains after cleaving the propeptide portion at one site. The process of maturation has been well investigated in the secretion of B. subtilis a-amylase. O h m u r a et al. (1983) have reported that the a-amylase gene of B. subtilis contains a region for a prepeptide (secretion signal sequence) and a short propeptide. They have suggested that the precursor of o~-amylase is processed by a two-step cleavage, removal of the secretion signal and removal of the propeptide, and that a certain kind of maturation enzyme other than signal peptidase involved in the deletion of the propeptide should be present. One possible explanation of our observation is that the prepeptide portion in the fused precursor of a-amylase could be processed by the maturation enzyme after the cleavage of the prepeptide by signal peptidase during secretion. It can be considered that the maturation enzyme can recognize the N-terminal of a-amylase and exactly cleave the N-terminal extension even though the artificial sequence is fused. In the case of h G H , the maturation enzyme involved in a-amylase secretion does not seem to function. The secreted h G H , however, did not contain the propeptide portion, suggesting that the propeptide portion can be processed even in the heterogeneous fusion during secretion by some enzyme. In the case of a-amylase, it is possible that the propeptide portion was processed by this enzyme at first and then the residual N-terminal extension was deleted accurately by the maturation enzyme for a-amylase. It is interesting how the propeptide portion fused to the h G H can be recognized and why the N-terminally truncated h G H could not be formed.

Acknowledgements We thank K. Yamane for plasmids, strains and valuable suggestions. We are indebted to R.F. Whittier for critical reading of the manuscript and discussions. This work was carried out as a national project 'Research and Development Project of Basic Technology for Future Industries' supported by the Ministry of International Trade and Industries.

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