Gene. 121 (1992) 111-119 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

111

037X-l 119/92/$05.00

06726

Secretion of human blood coagulation lipolyt ica (Yeast expression

vectors;

heterologous

Ckcile Tharaud a, Anne-Marie Ii Lahorutoire Received

de GCnPtique INRA-CNRS

by J.K.C.

Knowles:

rerouting;

transglutaminase;

clotting

cascade)

Ribet a, Claude Costes b and Claude Gaillardin a

and’

31 January

secretion;

factor XIIIa by the yeast Yarrowia

Laboratoire

de Chimie Biologique, Institut National Agronomique

1992; Revised/Accepted:

22 May/9 June 1992; Received

Paris-Grignon,

at publishers:

78 850 Thiverval Grignon, France

7 July 1992

SUMMARY

The industrial yeast, Yarrowia lipolytica, secretes high yields of an alkaline extracellular protease (AEP), which is synthesized as a preproprotein encoded by the XPR2 gene. We investigated the possibility of using this system for the secretion of human coagulation factor XIII subunit a (FXIIIa). This protein is naturally secreted in the plasma by an unknown, signal peptide-independent mechanism and has so far been found to be nonsecretable in yeast. We have designed six hybrid genes encoding fusion proteins between increasing portions of the AEP preprodomain and the precursor or mature forms of FXIIIa. All constructs directed translocation of the FXIIIa precursor into the endoplasmic reticulum. Transport of the translocated and core-glycosylated hybrid precursor to the Golgi apparatus appeared to be strongly rate limiting, and most of the precursors appeared to be partially proteolysed. One of these constructs directed the extracellular secretion of a low amount of hyperglycosylated FXIIIa. These results indicate that fusion to the yeast AEP signal peptide and dipeptide stretch allows FXIIIa to be translocated, albeit inefficiently, through the endoplasmic reticulum and to follow a classical secretory transit.

INTRODUCTION

Factor XIIIa (FXIIIa) is the catalytic part of human blood coagulation factor XIII, the last zymogen to become

Correspondence

PG-CBAI,

to: Dr.

C. Gaillardin,

78 850 Thiverval

Fax(33-1)

Laboratoire

Grignon,

France.

de Gtnttique,

30 81 54 57. A, absorbance

Abbreviations: extracellular

protease;

(1 cm); aa, amino acid(s);

Ap, ampicillin;

bp, base pair(s);

linked immunosorbent assay; EndoH, endoglycosidase XIII; FXIIIa, FXIII subunit a; FXIIIa, gene encoding terferon;

INA

Te1.(33-1) 30 81 54 52;

kb, kilobase

ribonucleotide;

PAGE,

merase

reaction;

chain

or 1000 bp; nt, nucleotide(s); polyacrylamide-gel PMSF,

AEP, alkaline

ELISA,

oligo, oligodeoxy-

electrophoresis;

phenylmethylsulfonyl

enzyme-

H; FXIII, factor FXIIIa; IFN, inPCR, fluoride;

polyS.,

Saccharomyces;

activator; extract/2”, 6.8.

SDS, sodium dodecyl sulfate; tPA, tissue plasminogen XPRZ, gene encoding AEP; Y.. Yarrowia; YPDm, I”,; yeast glucosej59,

Difco Bactopeptone/50

mM phosphate

buffer pH

activated during the clotting cascade. Thrombin-activated FXIIIa (FXIIIa*) is a transglutaminase (EC 2.3.2.13) which promotes the stabilization of the fibrin clot: it catalyzes the formation of covalent intermolecular y-glutamyl&-lysine bonds between fibrin CIor y chains (McDonagh, 1987). Factor XIII congenital disorders (Girolami et al., 1991) or partial acquired deficiencies in patients suffering from severe illnesses will provoke lifelong abnormal bleeding tendencies, lethal intracranial hemorrhages, abnormal wound healing, spontaneous abortion. Up to date, replacement therapy consists of monthly injections of placental FXIIIa concentrate (Rodeghiero et al., 1991). Since it is free of blood contaminants, recombinant FXIIIa may be valuable both as a substitution product and as a component of surgical glues (Lindsey et al., 1990). Recombinant FXIIIa would also allow food texturation during fermentation processes, through reticulation of proteins such as

112 myosin, caseins, or soya globulins (Traore and Meunier, 1991) and in this regard, secretion of even low levels of FXIIIa by a suitable microorganism may be useful. Authentic recombinant FXIIIa has already been obtained as a cytoplasmic protein in the yeast S. cerevisiae (Bishop et al., 1990b; Jagadeeswaran and Haas, 1990; Rinas et al., 1990), but attempts to have it secreted by yeast remained unsuccessful until now (Bishop et al., 1990a). Several aspects of FXIIIa biosynthesis remain obscure, in particular its secretion process. The FXIIIa homodimer does not reach circulating plasma after a classical secretory transit (Muesch et al., 1990). Molecular analysis revealed that its primary sequence shows no N-terminal nor internal signal sequence; that no carbohydrate is attached to any of its six potential N-glycosylation sites; that its N-terminal Ser residue is acetylated; and that none of its nine Cys residues is involved in a disulfide bond (Grundmann et al., 1986; Ichinose et al., 1986; Takahashi et al., 1986). These features are consistent with a cytoplasmic localization, and do not provide any clue on the natural secretion pathway of factor XIIIa. The yeast Y. Zipolytica presents a great potential for heterologous protein secretion. It naturally secretes an alkaline extracellular protease (AEP), encoded by the XPR2 gene, at levels up to l-2 mg/ml at high cell densities, and has been used to direct successfully the production and secretion of bovine prochymosin, S.cerevisiae invertase, porcine al-interferon, and human tPA (Buckholz and Gleeson, 1991; for a review see Heslot, 1990). AEP is initially synthesized as a large precursor with a 1%aa signal peptide, a stretch of nine X-Ala/X-Pro dipeptides (Ala-Pro-LeuAla-Ala-Pro-Ala-Pro-Ala-Pro-Asp-Ala-Ala-Pro-Ala-AlaVal-Pro), a 122-aa propeptide followed by a Kex2-like cleavage site, and a mature part (Matoba and Ogrydziak, 1989). We show here that fusion to the pre-(X.Ala/X.Pro) domain of AEP allows the secretion of FXIIIa into the growth medium.

RESULTS

AND DISCUSSION

(a) FXIIIa cDNA FXIIIa cDNA human placental Alto, CA) with

cloning clones were obtained by screening a lgtl 1 library (Clontech Laboratories Inc. Palo three 32P-labeled oligo probes designed

a All oligos were synthesized under each oligo indicates

with a Cyclone whether

DNA synthesizer

its sequence

reproduces

(Biosearch). the mRNA-like

from published nt sequence of FXIZZa cDNA (Grundmann et al., 1986; Ichinose et al., 1986) (for oligo sequences, see Table 1). The frequency of positive clones (3 out of 150 000 phages screened) was consistent with published data concerning immunoscreening of this expression library (Ichinose et al., 1986). The three inserts were subcloned in pBR322 as BamHI fragments; restriction analysis and comparison with published data showed that the longest insert was nearly full length. Sequence analysis revealed that the cloned FXZIZa cDNA insert lacked the 5’ noncoding region and the first five nt encoding the start codon and part of the N-terminal Ser’ codon. The rest of the sequence up to the RglII site (nt = 2814) was strictly identical to that published by Ichinose et al. (1986). (b) XPR2-FXZZZahybrid genes design and assembly; the (pre-X.Ala/X.Pro),,, directs the secretion of FXIIIa To direct the secretion of FXIIIa via the secretory pathway, we fused increasing lengths of the XPR2 region encoding the AEP prepropeptide to cDNA fragments encoding either the zymogenic form of FXIIIa or the activated form of FXIIIa, FXIIIa*. Since the 3’-untranslated region of FXZIZa cDNA was seen to affect negatively protein production in Y. lipolytica (not shown) as already reported in S. cerevisiae (Bishop et al., 1990a; Broker et al., 1991) it was deleted from the cDNA clone. The reconstituted shortened cDNA was inserted downstream from the XPR2 promoter and preprosequences, and upstream from a minimal XPR2 terminator sequence to generate pINA (see Fig. 1). Deletions in the preproregion were generated in vitro and inserted in pINA between the BglI or AvaI, and SmaI sites (Fig. 1) so as to abut the N-terminus of FXIIIa to the AEP signal sequence (Ala15; p-13a), to the dipeptide stretch (Pro’3; pD-13a), or to the entire prosequence (Arg15’; pDP-13a and pDP-13a*). The cDNA fragment encoding activated FXIIIa (FXIIIa*) was only fused behind the entire XPR2 preprosequence, so that no mature enzyme should be generated in the secretory pathway before the late Golgi apparatus, where KexZ-like proteolytic processing was expected to occur. All junctions between the XPR2 preprosequences and FXlIIa cDNA were generated by PCR so as to generate precise fusions and to reconstitute the 5’-terminal coding sequence of the FXZIIa cDNA (see Table I for the fusion sequences). A few silent mutations Their sequences (rightward)

are given from 5’ to 3 ’ , except for LA2 and LA4. The arrow

or complementary

(leftward)

strand

of the corresponding

gene.

(1) indicates the fusion site between the XPR2 and FXIIIa sequences. The aa sequences are indicated under each fusion oligo, from N- to C-terminal. Small letters indicate mutated nucleotides. Screeningprobes. Oligos were derived from the FXIIIu published sequences (see section a). Linkers. LAI and LA2 were used to construct pINA (section b and Fig. l), LA3 and LA4 to generate the pDP-13a* fusion (section b and Fig. 2). LA 1 and LA2 inserted six mutations

in the XPR2 sequence

in order to create

a Sfl site; three of those introduced

an Ala codon

replacing

Asn”‘.

The original

AsnlSJ

was restored by cloning the LA3-LA4 linker into pINA cut by Sfi + &a1 to create the pDP- 13a* fusion (see Fig. 2). PCR primer.\. The primers V.VC used to create the different fusions shown in Fig. 2. TC15 and TC16 were designed to bring additional restriction sites (Sfil and Yhol).

113 TABLE

I

Oligos used for hybrid genes construction

Screening

CT1 GAC +

ACC

*

AGC AAA

AAC

CCA GCA TTG

GGC

(FXlllo....Gly”‘)

probes CT2 GGA GGA GAT +

CT3 GAC +

GGC ATG

CCA GGG CCC

ACT CAT

GCA CAC TTC

I-W

CCA CTG CAC

CC8 GCc TCT

TCg 8cc GCC AAG

CGA

GQ

AGc ceg CGG TTC

GCT

CGe AGA

(XPRZ .. Lys CT AAT GA AGA TTA

A

Tz

II Ii

KpnI

CCCGGGTGGACGTCTAGAGGTAC LA2 c

GGGCCCACCTGCAGATCTC

Argls’//

polylinker)

GCC AAG C CGG TTC

G

LA4

(XPR2....Lys’““)

PCR

XbaI

SmaI

SfiI ATT

C TAA

LA3 +

ACT TAC

(FXllh....Val6’0)

LA1 CC GAG -

ATT

(FXIIh...Tyr’s’)

A vaI

Linkers

ATG GAT

-

va1

CTC CCC

ATT

CCT

GCT TCT

TCT

AAT

GCC AAG

CGA

IlTCt

GA8 ACT TCC AGG

ACC GCC ‘ITT

Lys Arg’“‘//Ser~. . . FXllln)

(XPRZ...

primers

EglI Tz

ACT

GCC GTT CTG GCC GCT CCC CTG GCC (XPR2..Aln’9) GGC

TC5 TCC +

AAA

TC6 CTC +

ACT a

TC8 AAG +

AAA

GGT CCT GGA AGT

cTC get

GTT

CTG GCC

IiTCt

II AGG

CAC AGC

AGC AGG

GGC AGC

ATC

Pro’=// Se+. . . FXIIIa)

(XPRZ......

GA8 ACT TCC AGG

ACC GCC TTT

GGA

(XPR2 . . . . . Al~~“//Serl...FXllI~) CTC TTG

CAG

GTT GAC GC (nt 200 of FXIIIa

sequence,

reverse complementary

TC12 GGCAAACTATCTGTTAATTGCIITCA CAT (XPR2 Terminatorl/Stop..EXIllo)

CGA

AGG TCG

(reverse

TCT

strand) TTG

complementary

AAT strand)

XmnI TC13 CCA

ATG

AA-

ATG T’I% CGT (FXIIIa...Arg6s*)

BglII ‘24

No11

GCTGCAAGATCTGCGGCCGC

GCCACCTACAAGCCAGA

(nt 2382 of XPR2

reverse complementary BglI TClS -

CTC

Tz6

CTC

ACT

GCC GTT

SfiI CTG m

GCc CCC CTG CCC/l

sequence,

strand)

XbaI TCt GA8 ACT

TCt

ACC

GCC TTT

(XPRZ ..,, A Ials// Ser*. ,. FXlIla) BglI ACT

U

XbaI

GTT CTG CCC

GCT CCC //TCt GA8 ACT TCt

(XPR2...Pro~~llSer~...FXIlIa)

ACC GCC TTT

GGA

GGA

GGA

114 EcoRI

EcoRI Hind111

Hind111 I I he1

AvaI StiI SmaI XbaI KpnI

pINA (8.7 kb)

Eco

Minimal XPRZ terminator

Sal1

Not1

ti

EcoRI

Hind111

m

XPR2 promoter AEP prodomain

El

FXIIIa domain

0

Marker gene

pINA (7.1 kb)

FXIIIa* Fig. 1. Construction EcoRl-BglII a pBR322 fragment

of FXIIIa

fragment derivative

pINA502.

We cloned

with a 3’ Not1 restriction (Nicaud

This construct

was then fused downstream Briefly, pINA

sequence

plasmid

fragment

was cloned

placental

and 534 bp of the 3’ flanking Separately,

[from XmnI (nt = 2120) to stop codon]

into pINA366.

from the XPR2 promoter

sequence, replacing

library

(see section

a). An

region, Lvas subcloned

we synthesized

into

by PCR a XmnI-Bg/II

fused to a short XPR2 terminator

to Yon and Fried (1989), using pINA

as the polymerization

(130 bp

malrix for the

with TC12 as fusion oligo. and TC I3 and TC 14 the 3’ untranslated

and preprosequences

all the 5’XPRZ prepro coding sequences,

contains

from a igt I1 human

was called pINA366.

et al., 1989) as the matrix for XPR2 3’-flanking

(see Table I). The resulting

elsewhere).

fragment

except the two first codons

site. This was done according

PCR primers

be described

an FXIIIa cDNA

sequence,

a Bg/II site in place of PvuII. The resulting

FXIIIa 3’ coding region, pINA as external

vectors.

the last 160 bp of the FXIIIa coding

which carried

from stop codon)

secretion

the entire FXIIIa coding

carrying carrying

BamHI

region of F,%‘IIIu. This yielded

derived from the expression

the LAl-LA2

oligo adaptor

vector pINA

(to

(see Table I) inserted at the AwI

site (nt 2537) present at the end of the AEP prosequence, encoding

the AEP prodomain,

tween the EcoRI-SmrrI

fragment

with one substituted from pINA476,

followed by a synthetic XPR2 terminator. This linker reconstitutes the 3’-end of the XPR2 sequence aa (Asn ‘s4); it also brings three unique cloning sites: .SfiI, SmaI, and XhaI. A three way ligation bc-

SmaI-BglII

fragment

from pINA50,2

and BglII-EcoRI

fragment

from pINA

yielded pINA503.

This

plasmid was then used as the recipient for all the PCR synthesized XPRZ-FXIIIu fusion fragments described in Table 1, to generate a series of plasmids which carried the hybrid cassettes represented on Fig. 2, under the transcriptional control of the complete XPR2 promoter and of a minimal XPR2 terminator and excisable by a M/u1 + Nor1 digestion. To insert these cassettes into yeast integrative or replicative vectors carrying selcctablc markers. wc constructed pBR322 derivatives carrying a Not1 linker al the PvuII site, a Mu1 linker at the BumHI site, and the 5’ upstream region of XPR2 as a HindIII-M/u1

fragment

inserted between Hind111 and MuI. As required, we introduced

et al., 1989) or LEU2 as an EcoRI fragment sued from pINA plasmids,

(Fournier

from pINA

(Gaillardin

et al.. 1991): see, for example,

so that each of the fusion genes could be expressed

3’ sequences flanking the XPR2 coding scquenccs arrow, marker genes as large dotted arrows.

are depicted

pINA437.

URA3 between the EcoRI and Hind111 sites (from pINA 156; Nicaud

and Ribet, 1987) and/or

finally the I’.

The M[uI-Not1 XPRZ-FXIIIa hybrid

in an integrative

or replicativc

context,

as thin black boxes, preprosequences

together

lipo&ica

cassettes

ARSIB as a RUMHI fragment were cloned

is-

into each of these

with LEU2 or URA3 markers.

The 5’ and

as thick black boxes, FXIIIu sequences as an open

115 were introduced in the FXIIIa 5’ coding region, in order to establish a more favorable codon bias at the fusion site (Heslot, 1990). After insertion into pINA503, the hybrid constructs were excised as MuI-Not1 cassettes, and were transferred into yeast transforming vectors as described in Fig. 1. The resulting plasmids were used to transform Pold, a Y. lipolytica uru3leu2xpr2 strain, by the lithium acetate procedure (Gaillardin and Ribet, 1987). This strain (to be described elsewhere) is a derivative of the industrial strain W29 (ATCC20460) where non-reverting mutations were created by transformation. Integrative plasmids were linearized by Mu1 digestion to target the constructs to the XPR2 chro-

Em

AEP signal

ml

AEP X-Ala/X-Pro

a3

AEP prodomain

sequence

I

FXIII prodomain

I

AEP or FXIIIa

+

Known

Puten,ia,

+

Fig. 2. Schematic

stretch

mature domain

glycnsylation

site(s)

of the XPR2-FXIIIa

representation

The first two boxes represent the sequences .4EP precursor (pDP-AEP) and the zymogen indicate

N-glycosylation

cellular transit osylated

in human

pDP-13a,

plasma

while none of the FXIIIa

samples

the constructs

pDP-13a*,

respectively, the (13a). Diamonds

sites: the AEP site is glycosylated

(large diamond),

boxes represent

hybrid constructs.

encoding, of FXIIIa

pD-13a,

(small diamonds).

encoding

AEP-FXIIIa

p.2d-13a,

p.ld-l3a,

during intrasites are glyc-

The following secretory and p-13a,

six

fusions: where p

stands for the AEP slgnal sequence, D for the AEP X.Ala or X.Pro dipeptidc stretch, 2d for the first two dipeptides of the former stretch, Id for the first one, P for the AEP prodomain, and 13a* for the sequence

encoding

13a for the FXIIIa

activated

FXIIIa.

sent the oligoa which were used for the construction

sequence,

The arrows

repre-

of the hybrid genes.

LA oligos form a fusion linker, TC oligos were used as PCR primers (see Table 1). The pDP-13a, were generated polymerization

p.2d-13a.

and p-13a junction

and (TC6 + TC8) respectively. and

as the pairs of primers.

Primers were used

1 1 min at 72’C were carried out. The gent fusions beof

I PM. Twenty cycles of 0.25 min at 94’C,

tween the 3’ end of FXIlIrr

and XPR2 terminator

pD-13a fusion were obtained

according

matrices

fragments

using IO ng of pINA (see legend to Fig. I) as the matrix and (TC2 + TC8). (TC15 + TC8), (TC16 + TC8)

at a final concentration min at 55’C

p.ld-13a

PCR reaction.

(see Fig. 1) and the

to FXIIIu downstream

SnzaI site (nt 196). so that this site was included

from the

in each of the XPR2-

FX1lltr joins. TC2 carried

TCl6

and TC6 carried

was thus cloned while pD-13a.

the XPR2 .4wI site (nt 2537). and TC4, TC15, the XPR2 Bg/I site (nt 2138). The pDP-13a joint

into pINA

p.2d-13a,

p.ld-l3a.

(c) Influence of the AEP signal peptide on FXIIla secretion As mentioned above, the hybrid gene encoding p-13a did not direct the secretion of FXIIIa, whereas that encoding pD-13a did. We hypothesized that fusing of the FXIIIa hydrophilic N-terminus right after the last residue of the AEP signal peptide (Ala”) in p-13a might interfere with signal peptide recognition and/or cleavage. To test this

to Yon and Fried (1989) by a two

TC5 was used as the fusion oligo and TC4 and

TC8, as external primers. TC8 hybridized

mosomal locus of the recipient strain. Replicative plasmids carried the Y. lipolytica autonomously replicating sequence ARS18 which, exhibits centromeric functions and results in stable, low-copy-number plasmids (Fournier et al., 1991). All transformants were checked either by Southern blot analysis of chromosomal DNA for integrants, or by restriction analysis of reextracted plasmids for replicative transformants (Fournier et al., 1991). In order to identify the constructs which could direct the secretion of FXIIIa into the growth medium of transformed yeasts, stable Pold integrants were grown under XPR2 inducing conditions (YPDm medium) at 18°C. Samples were collected during early stationary phase when AEP production in a wild-type strain reaches a plateau (Nicaud et al., 1989a). Supernatants were concentrated by ultrafiltration and aliquots were deglycosylated using EndoH. Crude and deglycosylated samples were subjected to SDSPAGE and immunoblotting using anti-FXIIIa antibodies. Results presented in Fig. 3 show that the hybrid gene encoded by pD 13a directed the secretion of an immunoreactive product (compare lanes 1 and 4, 2 and 5). In contrast, none of the other constructs led to the secretion of an FXIIIa-immunoreactive material (data not shown). Results concerning the secretion of pD 13a protein are dealt with in section e.

(see Fig. 4) as an AvaI-XmuI

fragment,

and p-13a joints were cloned as BgII-

XnzaI fragments. analysed

After fragment

by restriction

figure is not drawn alternance dipeptides;

enzyme

insertion

into pINA503,

digestion

and dideoxy

to scale. The AEP signal peptide

all fusions were sequencing.

This

is 15 aa long; the

of black and white boxes represents the AEP series of nine the AEP prodomain contains 124 aa, the AEP mature part 297

aa: the FXIIIa part, 694 aa.

activation

peptide contains

37 aa and the FXIIIa

mature

116

A

pD-13a

T

T

ARS18

13a

---II

_ -------

_

+

+

-

kDa

lP;,

13a kDa

v~~~~-

+

~~~,#1

B

“”

-

91.4

-

69

-

46

I

200

‘ii

4,

-

12

97.4

34

B T

-69

56

7

pF3;-

PD13a

13a

8

9

10

11

pDP13a*

La

kDa --B---B----

-

123

4

Fig. 3. FXIIla natants

secretion.

(pD-13a) or from a replicative control

strain

(T). Crude

expressing

(- ) and

the analysis

pD-13a

(pD-13a/ARS)

for each strain. Placental

-

200

-

97.4

-

69

-

46

_.“I-‘--

,,__

_^_

67

The figure only presents strains

from the Leu’

compared

5

-

plasmid

EndoH-treated FXIIIa

of super-

from an integrated and from the Leu + (+ ) samples

were

(25 ng) purified from Hoechst

Fibrogammine (Traore and Meunier, 1991) was loaded as a control (13a). All strains were grown at 18°C on YPDm inducing medium as previously described (Fabre et al., 1991). Samples were collected ture, at a cell density of &,,,“,,, of 18 units, and PMSF centration),

and aprotinin

centrifuged

at 12000 xg for 10 min at 4°C.

EndoH

treatment.

ice-cooled, PMSFjS

and

l/l000

v/v) were added.

Samples were adjusted then

adjusted

mM Na.azide.

37°C after the addition or deglycosylated through

(Sigma;

to 50 mM

Millipore Ultrafree-MC

on 0. I “/b SDS/7.5%

polyacrylamide

teins were transferred membranes Diagnostica; anti-goat

to nitrocellulose

were probed I:1000

antibody

pH

5.512 mM

(Genzyme).

Then 400 ~1 crude

were submitted

to ultrafiltration concentrated

sample buffer. Aliquots were subsequently

(Schleicher

and an alkaline

(50 ~1,

fractionated

gels. After electrophoresis,

with a goat polyclonal

dilution)

(Biosys;

Na,.citrate

filter units 30000 NMWL,

to 200 ~1 culture supernatant)

to

out for 2 x 8 h at

to 50 pl, and diluted in 50 ~1 2 x Laemmli equivalent

Samples were

were subjected

was carried

of 1 mU of EndoH aliquots

Aliquots

123

to 0.1% SDS, boiled for 3 min,

Deglycosylation

supernatant

after 41 h of cul(2 mM final con-

the pro-

and Schuel), and the anti-FXIII

(American

phosphatase-conjugated

1:800 dilution).

possibility we generated two constructs (p.ld-13a and p.2d-13a, see Fig. 2) carrying one or two X-Ala/X-Pro dipeptide(s) after the signal peptide cleavage site, so as to possibly restore a suitable structure around the cleavage site, without introducing the whole dipeptide stretch. According to von Heijne’s rule (von Heijne, 1986) signal peptidase cleavage was predicted after Alal (not shown). However, none of these extended signal peptides directed secretion of FXIIIa (data not shown).

Fig. 4. Western

4

5

blot analysis

67

8

of FXIlIa

9

10

intracellular

11

accumulation.

We

crude ( - ) and EndoH-treated (+ ) samples of the Leu + conexpressing the trol strain (T), and of the Leu * integrative transformants

compared

constructs FXIIIa

indicated

on top of panel

(25 ng) purified from Hoechst

A and panel

B. (13a), placental

Fibrogammine

(Traore

and Meu-

nier, 1991). Cells were grown

and collected as described in Fig. 3. Cell pellets equivalent to AbOOnmof 4 were lysed in 80 ~11Laemmli sample buffer, heated at 95°C for 5 min, and disrupted by vortexing with 0.3 g of acid-washed

glass beads (0.45 mm). Then 320 ~1 of 1 x sample buffer

were added, and samples were boiled for 5 min; extracts equivalent

to AhOOnmof 0.6 were analysed

as described

issued from cells in the legend to

Fig. 3.

The four strains encoding p-13a, p.ld-13a, p.2d-13a and pD-13a were analysed for their intracellular content of immunoreactive FXIIIa. Crude and EndoH-treated cell extracts were subjected to immunoblotting with anti-FXIIIa antibodies after electrophoresis under denaturing conditions (Fig. 4A). The AEP signal peptide alone (p-13a construct) led to a low production of precursors as compared to pD-13a (Fig. 4A, lanes 3, 4 and 9, 10; Figure 4B, lanes 6, 7. and 8,

117 9). The p-13a products into the endoplasmic

were at least partially translocated reticulum, since treatment with

EndoH resulted in a mobility shift from approx. 100 kDa to approx. 88 kDa. This was consistent with removal of six N-linked core-carbohydrate chains. The slight difference observed between native FXIIIa (84 kDa) and these EndoH treated precursors (88 kDa) was not due to interfering yeast protein (native FXIIIa migration was not disturbed by addition of an equivalent amount of yeast protein, not shown). It may reflect residual N-acetyl glucosamine moieties or O-glycosylation, or retention of (part of) the AEP signal peptide due to impaired signal peptidase cleavage (see above). EndoH-treated cell extracts of the strain expressing the (secreted) pD- 13a construct exhibited a single 92 kDa band, suggesting that the hybrid protein was translocated and core glycosylated, possibly at all six Asn-X-Thr/Ser sites present in the FXIIIa zymogen (see Fig. 2). All sites however did not appear to be recognized and glycosylated at similar rates, since native samples revealed precursors of 102, 106 and 110 kDa (independent experiments, not shown). The different mobility of the deglycosylated precursors accumulated by strains expressing p-13a and pD13a suggested that the latter was not processed by dipeptidyl-aminopeptidase. We also examined the intracellular content of the strains encoding the two intermediate constructs. The p.ld-13a precursor accumulated as a glycosylated but degraded product of 58 kDa, bearing 6 kDa of N-linked core carbohydrate chains (Fig. 4A, lanes 7, 8). We have not elucidated whether this form was entirely translocated inside the endoplasmic reticulum or if it remained partially exposed to a putative cytoplasmic degradation. The expression of p.2d-13a precursor could not be detected (Fig. 4A, lanes 5, 6). Taken together, these results suggested that the signal peptide of AEP alone was able to direct the translocation of FXIIIa into the endoplasmic reticulum, but that the whole dipeptide stretch was important for FXIIIa secretion outside of the cell. (d) Role of the AEP propeptide in the secretion of FXIIIa The construct pDP-13a, with an entire AEP preprosequence, did not direct the secretion of FXIIIa. The immunoblot analysis of cell extracts revealed intracellular accumulation of glycosylated precursors. Before treatment with EndoH, we observed a major polypeptide of 72 kDa, and a minor one of approx. 90 kDa (Fig. 4B, lane 4). Removal of N-linked carbohydrates resulted in a 6-kDa decrease of both precursors yielding two products: a major one migrating faster than native FXIIIa (around 66 kDa), and a minor one with a mobility similar to that of FXIIIa (Fig. 4B, lane 5). This indicated that the precursors issued from pDP- 13a

expression were translocated, core-glycosylated, but that most of them underwent aberrant proteolytic cleavage. The amount of precursors accumulated was lower for pDP-13a* than for pDP-13a (Fig. 4B, compare lanes 4, 5 and 10, 11) but they similarly underwent limited degradation. A single 68-kDa product could be observed in the crude extract, which was reduced to 62 kDa following deglycosylation (vs. 80 kDa for FXIIIa*). The 4-kDa difference between this precursor and the 66 kDa of the major deglycosylated form issued from pDP-13a reflected the absence of the 4-kDa FXIIIa activation peptide. In summary, the presence of the whole AEP proregion within the hybrid precursors seemed to impair secretion and to promote aberrant proteolytic processing. (e) Secreted factor XIIIa As discussed above, the construct encoding pDP-13a was the only one to direct secretion of an immunoreactive product, although the efficiency of this process was low. Western blot analysis of supernatants concentrated by ultrafiltration showed a hardly detectable smear corresponding to a heterogeneous product composed of high M, species, while EndoH-treated samples allowed detection of a diffuse smear around 84 kDa (Fig. 4). Smearing after EndoH treatment may be due partly to the high amount of peptone present in concentrated YPDm samples, although attempts to purify the product could not rule out that the secreted product itself was heterogeneous (see below). We feel it unlikely that the extracellular accumulation of FXIIIIa related products could reflect cell lysis, since no trace of the intracellular form of 92 kDa was recovered outside of the cells. This rather suggested that passage through the Golgi apparatus resulted in secretion of a hyperglycosylated protein. We compared the secretion of FXIIIa-related products by strains carrying the hybrid construct either integrated at the XPR2 locus or on a replicative plasmid, with LEU2 or URA3 as selective markers. A slight increase in the amount of secreted immunoreactive material was observed with replicative plasmids as compared with integrated plasmids (Fig. 3). This reflected the high stability but low copy number (1 or 2 per cell) of plasmids carrying the Y. lipolytica ARS18 sequence (Fournier et al., 1991). No difference was observed in connection with the marker used (not shown). Temperature did not affect the level of FXIIIa secretion (data not shown). In order to quantify the secreted product, crude and deglycosylated supernatant aliquots were subjected to a double site ELISA, using a mouse monoclonal FXIIIaspecific IgG as the capture antibody and a rabbit polyclonal serum as the second antibody (isolation of monoclonal IgG specific for FXIIIa and development of an ELISA assay are to be published elsewhere). This assay indicated a pro-

118

duction of approx. 500-600 ng/ml for integrative or replicative pDP- 13a transformants, whereas control strains or nonsecreting transformants gave a lower than the threshold value of this test (5 ng/ml). In order to purify the hyperglycosylated secreted product away from the peptones, we concentrated a 2.5-ml aliquot of the supernatant from an integrative transformant through ultrafiltration followed by adsorption on concanavalin A Sepharose. The product was then eluted with x-D-methylmannopyranoside, subjected to EndoH treatment, SDS-PAGE, and immunodetection. The resulting immunoblot membrane exhibited a fuzzy band in the 85kDa range. The heterogeneity of the observed product may thus reflect heterogeneity of the secreted FXIIIa, due to limited proteolysis or other modifications such as O-glycosylation. This, however, confirmed that the amount of secreted FXIIIa did not exceed 1 pg/ml culture supernatant and precluded assessment of its transglutaminase activity. (f) Conclusions In this paper, we show that the signal peptide of AEP together with the X.Ala/X.Pro region can direct, albeit inefficiently, the secretion of human factor XIIIa in Y. lipo@tica. (1) Human FXIIIa is naturally secreted by an unknown, signal peptide-independent mechanism (Muesch et al., 1990). It is worthwhile noticing that FXIIIa expressed as a cytoplasmic protein, either in S. cerevisiae (Bishop et al., 1990a) or in Y.l@oLvtica (unpublished) is not spontaneously secreted outside the cells. We show here that FXIIIa can be secreted by the classical secretory pathway of Y. lipolytica, when expressed as an AEP-FXIIIa fusion precursor. To our knowledge this is, along with human interleukin lp (Baldari et al., 1987; Livi et al., 1991) the second example of a secretory protein without a classical signal peptide which has been rerouted through a yeast secretory pathway. Both interleukin l/j’ and FXIIIa underwent aberrant N-linked glycosylation. In the case of FXIIIa, transit through the secretory pathway resulted in additional modifications (perhaps limited proteolysis, O-glycosylation,...) which led to the formation of a heterogeneous product. (2) Whereas secreted FXIIIa was hyperglycosylated (see section e and Fig. 3), no hyperglycosylated form was detected within the cells: if undetected hyperglycosylated polypeptides had been present in the crude extracts, EndoH treatment should have enhanced the immunoblot signal, which was not the case (Fig. 4A, lanes 3, 4 and Fig. 4B, lanes 6, 7). This suggested that a rate-limiting step slowed down the transit to the Golgi apparatus, where outer-chain addition takes place, but that later steps were not rate limiting in the case of the secretable pD-13a construct. Nonsecretable constructs yielded proteins which were par-

tially degraded, probably inside the secretory pathway. This degradation may reflect early proteolysis of the heterologous hybrids or targeting to the vacuole and subsequent degradation. (3) The results presented here indicate that the whole prosequence of the XPR2 gene may not be useful for secreting heterologous proteins: it has no influence on the secretion of porcine IFN or bovine prochymosin (Heslot, 1990) and seems to hamper the secretion of FXIIIa. Interestingly, the prosequence of Kluyveromyces lactis killer toxin was shown to negatively affect the secretion of human serum albumin in S.cerevisiue (Sleep et al., 1990). Prosequences might be generally dispensable for the secretion of heterologous proteins, as already suggested by some reports on the prosequence of S. cerevisiae x-factor (Ernst, 1988). In the case of Y. lipolytica AEP, the prodomain was reported to be crucial for the intracellular transit of the protease precursor (Fabre et al., 1991). However, this function might be comparable to that of an internal chaperone and be specific for the AEP mature part (Fabre et al.. 1992). Whereas the whole prosequence seemed to be unnecessary or even deleterious for FXIIIa secretion, the signal peptide alone was not sufficient for extracellular secretion. Surprisingly, the AEP dipeptide stretch seemed to favor intracellular transit, as indicated by the results presented in section c. We have currently no explanation for the facilitating role of the dipeptide stretch in the secretion of recombinant FXIIIa, whereas its exact role during AEP secretion rcmains hypothetical (Kreil, 1990; Matoba and Ogrydziak, 1989).

ACKNOWLEDGEMENTS

We thank Philippe Joyet for the synthesis of oligos, Feng He for the PCR synthesis of the minimal XPR2 terminator used to construct pINA476, and Emmanuelle Fabre for the construction of pINA302. Special thanks to Christophe d’Enfert for helpful critical reading of the manuscript. Part of this work was supported by SOREDAB (Guyancourt, France).

REFERENCES Baldari, C., Murray, J.A.H., Ghiara, P., Cesareni, G. and Galcotti, C.L.: A novel leader peptide which allows efficient secretion of a fragment of human

interleukin

18 in Saccharwn~re.s

crrevisinr.

EMBO

J. 6

(1987) 229-234. Bishop, P.D., Teller, D.C., Smith, R.A., Lasser, G.W.. Gilbert, T. and Scale, R.L.: Expression, purification, and characterization of human factor

XIII

in Smchurmqces

wrevisim.

1861-1869. Bishop, P.D. and Lasser. G.W.. Trong,

Biochemistry

I.L.. Stcnkamp,

29 (1990a)

R.E. and Tcllcr.

119 D.C.: Human -

recombinant

crystallization

(1990b)

factor XIII from Saccharomyces

and preliminary

x-ray

data.

cerevisiue

J. Biol. Chem.

265

of the human

in Sacchuronzr~es host-vector

blood coagulation

cerevisiae: dependence

systems

and medium

of heterologous

M. and Amann,

protein

factor XIIIa

of the expression

conditions.

levels from

Appl. Microbial.

Bio-

Yeast systems for the commercial

proteins.

Bio/technology

9 (1991) 1067-

1072.

r-factor Fabrc,

and processing is mediated

cerevisiae

precursor.

E., Nicaud,

of heterologous

proteins

solely by the pre-segment

in of

Lopez,

extracellular

M.C. and Gaillardin, and secretion

proteasc.

C.: Role of the

of the Yarrowiu

J. Biol. Chem.

lipo/ytica

266 (1991)

3782-

3790. Fabre,

E., Tharaud,

C. and Gaillardin,

is rescued

C.: Intracellular

by transcomplementation

transit of a yeast

with its prodomain.

P., Guyaneux,

L., Chasles,

M. and Gaillardin,

ur.7 sequence isolated in a morphogenesis

C.A.,

Gorman,

W.D.,

Wilhelm,

M.C. and

using fibrin glut in a rat mastec-

J. and

A., Sathe, G.M.,

Young,

N-glycosylated

interleukin-lp

in Sacchuromycrs

leader

from

ulbicans.

peptide

Cundida

P.R.:

Simon,

Secretion

J. Biol. Chem.

of

using

cerevisiae

a

266 (1991)

Matoba, S. and Ogrydziak, D.M.: A novel location for dipeptidylaminopeptidase processing sites in the alkaline extracellular protease of McDonagh, (Ed.),

J. Biol. Chem. 264 (1989) 6037-6043.

lipobkcc.

J.: Structure Hemostasis 2nd

and function

of factor

and Thrombosis.

ed. J.B.

XIIIa.

Basic

Lippincott

In: Caller,

Principles

Company.

B.S.

and Clinical

Philadelphia,

1987,

pp. 289-300. Muesch,

A., Hartmann,

E., Rohde,

oport, T.A.: A novel secretory

K., Rubartelli, pathway

Gaillardin,

C.

and

/&galactosidase

Ribet, activity

l@o/~fica. Curr.

Girolami,

Genet.

A., Sartori,

factor

C.: Scarcity

A., Sitia, R. and Rap-

for secretory

protease (XPRZ) gene of the yeast 12 (1989) 285-298.

mutant of the yeast Yarrowiu expression

resistance

of

Ycrrrowia

coding

of

G. and Ktipper,

for human

factor

H.A.: Char-

XIIIa.

Proc.

Natl.

Sci. USA 83 (1986) 8024-8028. H.: Genetics

Yurroti,iu

and

genetic

Adv.

lipolyfica.

Rinas,

engineering

Biochem.

produced

proteins?

TIBS

Eng.

of the industrial Biotcch.

yeast

43 (1990)

43-

L.E., Fujikawa,

K. and Davie, E.W.: Amino

Jagadeeswaran,

P. and Haas,

P.: Synthesis

of human

coagulation

25

factor

XIII in yeast. Gene 86 (1990) 279-283. Kreil, G.: Processing of molecular

of precursors

ticketing.

TIBS

by dipeptidylaminopeptidases: 15 (1990) 23-26.

Yurrowiu

R., Broker,

G.: Characterization

in Socchcrromyces cereksiue. F., Tosetto,

pharmacokinetics

C.:

cxtracellular J. Biotechnol.

lipolJGu.

M., Kargcs,

H.E.,

ofrccombinant

Kilpper,

factor XIIIa

Bio/Technology

A., Di, Bona,

8 (1990) 543-

E. and Castaman,

of a placenta-derived

factor

type I and type 11 factor XIII deficiency. 30-34. Sleep, D.. Belfield, G.P. albumin

and Goodey,

from the yeast

different leader sequences. Takahashi,

N., Takahashi,

blood coagulation

acid sequence of the a subunit of human factor XIII. Biochemistry (1986) 6900-6906.

P. and Gaillardin,

of the alkaline

546.

strum

73. A., Hendrickson,

and amplification

U., Risse, B., Jaenicke,

Rodeghiero, classification

77 (1991) 565-569.

E., Zcttlmeissl,

of cDNA

in

11 (1987) 369-375.

Br. J. Haematol.

U., Amann,

acterization

directed

M.T. and Simioni, P.: An updated

XIII defects.

Grundmann,

LELI2

phleomycin

J.M., Fournier,

of

sequencing

H.A. and Zettlmeisl,

A.M.: and

Nicaud, J.M., Fabre, E., Beckerich, Cloning,

Yeast 7 (1991) 25-36.

lipo!viicu.

Ichinose,

Meyers,

J.

Biol. Chem. 267 (1992) 15049-15055. Fournier,

Heslot,

Spotnitr,

prevention

15 (1990) 86-88.

protease

Acad.

P.L.,

Practice,

DNA 7 (1988) 355-360.

J.-M.,

pro region in the production alkaline

T.M.,

Seroma

Livi, G.P., Lillquist, J.S., Miles, L.M., Ferrara,

Yurrowiu

Ernst, J.F.: Efficient secretion Saccharomyces

Masterson,

R.F.:

15348-15355.

technol. 34 (1991) 756-764. Buckholz, R.G. and Gleeson, M.A.G.: production

W.H.,

Morgan,

tomy model. Arch. Surg. 125 (1990) 305-307.

13888-13889.

Broker, M., Bauml, O., Gilttig, A., Ochs, J., Bodenbenner, E.: Expression

Lindsey,

human Traore,

placenta.

Am. J. Hematol.

A.R.:

Bio;Technology

Proc. Natl. Acad. J.-C.:

of human using five

cerevisiue

8 (1990) 42-46. F.W.: Primary

factor XIIIa (fibrinoligase.

F. and Mcunicr,

in

36 (1991)

The secretion

SacchuromJres

Y. and Putnam,

G.: Clinical

XIII concentrate

structure

transglutaminase)

of

from

Sci. USA 83 (1986) 8019-8023.

Cross-linking

of caseins

by human

pla-

cental factor XIIIa. J. Agric. Food Chem. 39 (1991) 1892-1896. von Heijne, G.: A new’ method

for predicting

signal sequence

cleavage

sites. Nucleic Acids Res. 14 (1986) 4683-4690. a case

Yon, J. and Fried, M.: Precise gene fusion by PCR. Nucleic Acids Res. I7 (1989) 4895.