AbstractImcpb, a gene from Leishmania mexicana that encodes a major cysteine proteinase in the parasite, has been cloned and sequenced. LmCPb is related more to cysteine proteinases from Trypanosoma brucel and Trypanosoma cruzi than to a previously characterized cysteine proteinase, LmCPa, of L. mexicana. It contains a long C-terminal extension characteristic of similar enzymes of T. brucei and T. cruzi. The gene is multi-copy and tandemly arranged. Imcpb RNA levels are developmentally regulated with steady state levels being high in amastigotes, low in metacyclic promastigotes and undetectable in multiplicative promastigotes. This variation correlates with and may account for the stage-specific expression of LmCPb enzyme activity

Coombs, Graham H.

Mottram, Jeremy C.

Souza, Augustine E.

Waugh, Sheila

Elsevier - Publisher Connector 

Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of Leishmania mexicana 

Volume 311, number 2, 124-127 FEBS 11642 
0 1992 Federation of European Biochemical Societies 0014S793/9?/$5.W 
Characterization of a multi-copy gene for a major stage-specific 
proteinase of Leishmania wrexicana 
October 1992 
cysteine 
Augustine E. Souza”, Sheila Waugh”, Graham H. Coombs” and Jeremy C. Mottram’ 
’ Wellcome ihil of Mokcuh Pamiroiogy, Insrirure of Getlerics, Uthersity qf Glasgow, Chrrclr Street, Glasgow Gil SJS, Scodatd, 
UK and ‘laboratory for Biochemical Pwasirofogy, Deparrmerrr of Zoology, Uuiversicy of Ghgow, Ghtsgow G12 8QQ, Scorkand, 
UK 
Received 1 September 1992 
btrqb, a gcnc from Leishntotriu rr~e.~icuna that encodes amajor cystcinc proteinasc in the parasite. has been cloned and sequenced. LmCPb is related 
more to cysteirre protcinases from Trypmosoma hrcei and Trypumsomrr crrr:i than to a previously charac~erizcd cystcine proteinase, LmCPa, of 
t, ttrrxicunu. It contains a long C-terminol extension characteristic of similar enzymes of ‘r. hrrei and ‘r. crlr5. The gene is multi-copy and tandemly 
arranged. hpb RNA levels are devclopmcntally rcgulaled with steady state levels being high in amastigotes, low in metacyclic promastigotes and 
undetectable in multiplicative promastigotes. This variation correlates with and may account for the stage-specific expression of LmCPb enzyme 
activity. 
Leiskrrwia msicarru; Cyst&c proteinase; Amastigote-specific; Developmental regulation 
1. INTRODUCTION 
Parasites of the genus Leishnania cause visceral and 
cutaneous disease in humans and other animals [l]. In 
its life-cycle, Leishnlaniu must infect two hosts, a mam- 
mal and an insect. The environments encountered inthe 
two hosts impose differing adaptive requirements on the 
organism and the three main forms of the parasite, the 
amastigote in the mammalian macrophage, the multi- 
plicative promastigote in the insect mid-gut, and the 
metacyclic promastigote in the insect mouthparts, are 
morphologically and biochemically very distinct [I]. 
One feature of the amastigotcs of L. nzesicurlu is the 
very high levels of cysteine proteinases (CPs) most of 
which are stage-specific and are thought to be involved 
in enabling survival within macrophages [2]. As part of 
our studies to assess the role of CPs in survival, and 
possibly virulence, we have cloned and sequenced a
cDNA encoding LmCPb, a CP of L. tnexicma primarily 
expressed in amastigotes [3]. 
2. EXPERIMENTAL 
Details oftheculturc and hnrvcstingofL. 11f. ~t~c.ricunu(MNYC/BZ! 
62/M379) and purification of L. mexicntra CPs are described elsewhere 
[4,5]. A 450 bp CP gene fragment was PCR-amplified from L. rrwxi- 
cam genomic DNA by using two degenerate oligonucleotide primers: 
no. I (5’ CCGAATTCCARGGICARTGYGGIWSITGYTGG 3’); 
and no. 2 (5’ CCAAGCTTCCAISWRTTYTTI ACDATCCARTA 3’) 
which correspond to highly conserved regions ofeukaryotic CP genes 
Currrs~crr~&~~ce aridress: AC. ivioiiram, inslilurs or Genetics. Church 
Street, University of Glasgow, Glasgow Gl I SJS, Scotland, UK. Fax: 
(44) (41) 330-5994. 
124 
[6.7]. The fragment was cloned and sequenced and found to encode 
par1 of a CP gene different from another ecently identified L. trwxi- 
LWIN CP gene, BHL~,VN [4], A cDNA library of L. rwxirnrut amastigotes 
constructed in AZAP II (Stralagene) was screened with the cloned 
PCR fragmenl and scscral hybridizing plaques were detected. 
Phagemids from several positive plaques were rescued. subcloned and 
sequenced by lhc Sungcr melhod [S] with Scqucnasc (USB) using 
plasmid and single-stranded DNA as templates; both strands of the 
largest cDNA. pSWbla. were complelcly sequenced over the region 
encompassing the open reading frame. The DNA scqucncc (deposited 
in the EMBL nucleic acid database with accession umber 214061) 
was analysed using the University of Wisconsin GCG package [9]. The 
entire insert was labelled with [l’P]dCTP by random priming and used 
as described previously [4.6] to probe Southern blots of L. mexiccwt 
DNA digested with various restriction enzymes and Northern blots 
of total RNA isolated from logarithmic phase promastigotcs (which 
comprise mainly multiplicative promastigotes), tationary phase pro- 
mastigotes (which contain a high proportion of metacyclic promasti- 
gotcs), aud from amastigotrs. A IzP end-labeled oligonucleotide com- 
plementary to tbc small subunit ribosomal RNA (5’ TA- 
CAATGGTCTCTAATCATCITC 3’) [IO] was used to probe the 
Northern blot as a control for RNA loading. 
3. RESULTS AND DISCUSSION 
A 2.2 kb cDNA clone, pSWbl+ was isolated from an 
amastigote cDNA library by screening with a PCR frag 
men1 amplified using degenerate primers based on 
highly conserved regions of CPs. The cDNA was full- 
length since the 5’ end carried 14 nt of the splice leader 
sequence which is present at the S’ ends of nuclear 
mRNAs in Leishna/ziu [I I] and the 3’ end had a poly(A) 
stretch. The 5’ untranslated region (UTR) of the cDNA, 
defined by the sequence between the 3’ end of the splice 
leader and the first in-frame ATG codon, was 144 nt 
long, We did not sequence all of the 3’ UTR for 
Volume 311, number 2 FEBS LETTERS October 1992 
LmCPb 
TCCP 
TbCP 
LmCPa 
DdCPl 
HSCPL 
LmCPb 
TCCP 
TbCP 
LmCPa 
DdCPl 
HsCPL 
l.mCPb 
TCCP 
TbCP 
LmcPa 
DdCPl 
HsCPL 
LmCPb 
TcCP 
TbCP 
LmCPa 
DdCPl 
HsCPL 
LmCPb 
TcCP 
TbCP 
LmCPa 
5dCPl 
HsCPL 
LrnCPb 
TcCP 
TbCP 
LmCPa 
DdCPl 
HsCPL 
LrrtCPb 
TcCP 
TbCP 
-125 -100 . 
MATSRAALCAVAVVCVVLAA..ACAPA~AIHVGTPAAALFEEFKRTYGRAYETLAEEPQRLA 
MSGW-RALSL-A-L--M.-CLVP-ATASL-AEETL-S9-V---9KH--V--SA---RF--S 
NPR-EMVRFVRLP--LLAM--CL-SVALGSL--EESLEMR.~--KK--KV-KDAK--AF-FR 
MARRNPLLFAIV-TILF--CYGSALI-RTPPPYDNFV-S-HYGS--KRH-K-FGGD---GH-FN 
MK-ILLFVLAVFTVFVSSRGIPPE~RS9-L--PDKFNKK-.SHE-~LE-FE 
MNPTLl--FCL-GIASATLTFDHSLE-QWTKW-AMHN-L-.~N--GY-R- 
-5f 
NFERNLELMREHQARNPHAQFGIT.... KFFDLSEAEFMRYLNGAAYFAAAKRHMGHYRKARADLSA 
V-RE--F-A-L-A-A----T--V-...P.-S--TRE--RS--H----H----QER-RVPVNVEWG... 
A--E-M-QAKIPA-A--Y-T--V-...P.-S-MTRE--R---R---S-----QKRLRK~VNVTTGR.,. 
A-KQ-MPTAYFLNTQ----HYDVS...G--A--TPQ---KL---.PD-Y-RHLKNHKEDVHVDDS.... 
I-KS--GKIE-LNLIAINHKADTKFGVN--A---~--KNY---NKE..-IFfDDLPVADYLDDEFINS 
yW-K-M~IEL-NPEYREGKHSF-MAMNA-G-MTSE--RQV....MN..GFQN-KPRKGKVFPEPLFYE 
1 50 
. . 
VPD... AVDWREKGAVTPVKDQGACGSCUAiSAVGNIEGPWYLAGHELVSLSEQQLVSCD.......... 
RPA ..~.....~~....~.....Q......~..~..~.~..~....~.~~....~....~.__.______ 
100 
DMDNGCSGGLMLPAFDWLLPNTNGHLHTEDSYPYVSGNGYVP~CSNSSELVVGAQIDGHVLIGSSEKAMA 
KT-S--G ----NH--E-IV-EN--AVY -------A--E-IS-P-TT-GH.T---T-T---ELPPD-Aal- 
TI-S--N ----DN--N-IVNSNG-NVF--A---------~Q-Q-QHNGH.El--A-TD--DLPQD-D-I- 
NI-E--N----D--MN-IM-SH--SVF--A----T--G-TR-P-HDEG,.E---K-T-FLSLPHD-ERI- 
AC-E--N ---PPN-YNYIIK.. --GIQ--S----TAET-TQ.. -NFN-A.NI--K-SNFTM-PKN-TV-- 
PGNE--N ----DY--QYVQD..--G-DS-E----EA.TEES..-KYNPK.YSV-NDT-F-D-PKQ---LM 
150 
AWLAKNGPIAIALDA..SSFMSYK~GVL..TACIGKPLNHGVLLVGYDWTG.....EVPYWYIKNSWG~ 
.CV.V...V.V.V......N.T.YG..N...S.VSE..D........N~SA.,~._A....I.....YAQ 
.Y..E...L...V..,,~...D.NG.I.,..S.TS...D.....~..N~NS_..._NP...I.....SN~ 
E-VE-R- *V-V-V--. . TTWQL-FG--V..SL-LAWS------I--FNKNA.....KP---lV-----SS 
GYIVST--L ---A--VE..~F-IG--.FDIP-NPNS-D--I~i--.SAKNTIFRKNM---IV-----A- 
KAV-TV---SV-I--GHE-.LF--E-IYFEPD-SSEDHO----V--.GFESlESDNNK.--LV-----EE 
200 +I 
WGEQGYVRVVHd.VNACLLSEYPYSAHVRES~APGTSTSSElPAPRP...VWEQVICFDKNCRRGCRKT 
---D--I-IAK-.S-Q--VK-EAS--V-GGPGPTPED-TTT-TSAPGPSPSYFV-MS-T-AA-IV--ENV 
---D--I-IEK-.T-P--MNPAYS--V-GGPTP-PP?PFPPSATFT........-OF-EG-G-TK--SHA 
. ..K..I.LA.. .S-P-M-KN--.-, ?.ESPHTPH7-T-TA 
------IYLRR-.K-T-GV-NFVSTSII 
--HG---KNAKDRR-H-GIASAASYPT- 
50 100 . 
LIKANECHKNGGGGASHIKCSPQKVTHCTYS.NEFCVGGGLCFETHDGKCSPYFFGSIHNTCHY~ 
TLPTGQ-LLTTS-VSAIVT-GAETL-EEVFFTSTH-S-PSVRSSVPLN9-NWLLR--VEFF-GSS~~24) 
TFPTG--VPTT-V-SVIAT-GASNL-QlI-PLSRS-S-PSVPITVPLD--I-ILI--VEYH-STNC~l2~ 
Pig. I, Alignment of LmCPb with selected cyslcine proteinases. TcCP=7’. cnci (CYSP_TRYCR); TbCP=T. brucpi (CYSP_TRYBR); LmCPa=L. 
wuxicuna (LCPA_LEIME); DdCPl =Dic~ymrehm disroidcum CPI (CYSl_DlCDI); llsCPL=human cathepsin L (CATL_HUMAN). Annotations 
in brackets refer to accession ames in Swiss-Pro1 22. Numbers above the LmCPb sequence are described in the text. Dashes in other sequences 
indicate identities to LmCPb. Dots indicate gaps made LO maximize alignmcnrs. Doubly underlined residues in the mature regions ofsome sequences 
represent the N-terminal amino acids of the mature CP as determined by amino acid sequencing. 
pSWbla, but, as for several other mRNAs of Leishna- 
nict, the itncpb UTR appears relatively large (ca. 800 nt). 
The iargesr open reading frame predicted a443 amino 
acid, 47.8 kDa protein which was aligned to trypanoso- 
matid and other eukaryotic CP sequences using GAP [9] 
and manually (Fig. 1). From this alignment, and from 
N-terminal amino acid sequencing of L. mexicana CPs 
(see below), residues -125 to -1 of LmCPb would be 
expected to form the pre- and pro-regions which are 
post-translalionally removed to produce the mature 
125 
Volume 311, number 2 FEBS LETTERS October I992 
Table I 
Percent identity beween LmCPb and other CPs for the different 
domains 
TcCP 
TbCP 
LmCPa 
DdCPl 
HsCPL 
PrcIPro 
44 
36 
27 
20 
19 
Central 
59 
59 
56 
:: 
C-terminal 
22 
26 
proteinase with a predicted size of 34 kDa. Residues 
l-218 comprise the central domain of the proteinase 
while residues cl to i-100 make up a C-terminal exten- 
sion similar to those present in CPs of T. brucei (TbCP) 
[6] and T. crud (T&P) [12,13]. As summarized in Table 
I, LmCPb is more related to TcCP and TbCP than it is 
to LmCPa or the non-trypanosomatid CPs. 
The end of the central domain (defined in [2,4,6]) and 
the start of the C-terminal extension was defined by 
comparing the LmCPb sequence with the two other 
trypanosomatid CP sequences which possess lengthy 
extensions. In TbCP, a run of 9 Pro residues is associ- 
ated with the beginning of the extension and in TcCP, 
G consecutive Thr residues are present. In LmCPb, how- 
ever, the demarcation between the central domain and 
the extension is less clear: at the analogous position is 
a stretch rich in Ala, Gly, Pro, Ser, and Thr. The only 
other L. mexicana CP cDNA sequenced so far (Inzcya) 
lacks a comparable C-terminal extension but does ex- 
tend a short way beyond the Dictyosteliunz and human 
sequences with the extra amino acids again being pre- 
dominantly Pro and Thr [4]. Of the identities common 
to LmCPb, TcCP, and TbCP in the C-terminal exten- 
sion, 8 are Cys residues. An examination of the exten- 
sions of alpha- and beta-oryzains [14] indicate that 
‘they are also rich in cysteines but these residues do not 
appear align with those of LmCPb, TbCP, or TcCP. 
Potential roles for the extension include targeting, sta- 
bility, or interaction with protein substrates. 
The CP content of e. mexicuna is complex [2,3,15]. 
Amastigotes contain 3 main classes (A, B and C) that 
are distinguished on the basis of differences in apparent 
size, substrate specificity, and glycosylation [3]. Interest- 
ingly, direct amino acid sequence analysis of purified 
CPs of these classes yielded the same N-terminal se- 
quence for each (Robertson and Coombs, unpublished 
results). This sequence is identical to that present in the 
fmcpb open reading frame (Fig. 1). The observed iffer- 
ences among these E. rmximna CPs could be due to 
variable post-translational processing of a single CP 
gene product. For example, a potential N-linked glyco- 
sylation site is present at position 103 in the centm! 
domain where the consensus VPECSNSSELVVGA oc- 
curs: glycosylation at this site could account for the 
difference between types B and C CPs which do not bind 
126 
23.7 - 
9.7 - 
6.6 - 
4.3 - 
2.3 - 
2.0 - 
0.5 _ 
123456 7 
-23.7 
“I. 
-4.3 
-2.3 
Fig. 2. Cienomic organization of f~tzcpb: total gcnomic DNA (IO ,~g) 
was digested to completion with restriction enzymes, electrophoresed 
through 1% ngarose, blotted, probed with the pSWbla cDNA probe. 
Lane 1-6: EcoRI, Sull. SacI, HindHI, Xbul, BrrrnWI. Marker sizes arc 
shown on the left. Lane 7 shows a partial digest of total DNA digested 
with EcoRI and probed as above. 
to concanavalin-A Sepharose columns and type A CPs 
which do [3]. Alternatively, a number of closely related 
but distinct genes could exist. 
To characterize the genomic arrangement of hcpb, 
a Southern blot of total L. mexicana genomic DNA cut 
to completion with various restriction enzymes was 
probed with the hcpb cDNA (Fig. 2; lanes l-6). Three 
anrrymes (&RI, Sal1 and SKI) gave a major band at 
2.8 kb whereas 3 other enzymes (HindIlL XbaI and 
BantHI) gave a major band of ~23 kb. A blot of DNA 
partially digested with EcoRI gave a hybridization lad- 
der beginning at 2.8 kb (Fig. 2; lane 7) implying that 
fmcpb is a multicopy gene of 2.8 kb unit size with at least 
10 tandemly repeated copies per locus. The minor bands 
seen in some of the digests may represent flanking se- 
quences, polymorphisms or possibly copies located at 
other loci. This organization in which the majority of 
genes is present as a tandem array is similar to that 
reported for the T. brucei CP [6] and T. cruzi CP [ 12,131 
Volume 311, number 2 FEBS LETTERS October 1992 
1 2 3 
9.5 _ 
Fig. 3. Developmental differences infmcpb RNA levels: Tenpg of total 
RNA from multiplicative promastigotes (lane I), stationary phase 
promastigotes (lane 2) and amastigous (lone 3) were probed with the 
i~trcpb probe (upper panel) or with an oligonuclcotidc complementary 
to the small subunit rRNA (lower panel). BRL RNA ladder sizes arc 
shown on the left for the upper panel. 
but different from that of the other L. mexicana CP 
gene, I~ncpa, which exists as a single copy gene [4]. 
The hncpb cDNA was also used to probe Northern 
blots of total RNA from the three major developmental 
stages of the parasite (Fig. 3). Strong hybridization was 
seen for the fnzcpb RNA from amastigotcs at 2.3 and 
weaker signals were seen at -5 and -8 kb. A weak signal 
at 2.3 kb was detected in metacyclic promastigote RNA. 
fmcpb RNA was undetectable in multiplicative promas- 
tigotes. The 2.3 kb transcript corresponds well with the 
size of the cDNA and the larger signals could be the 
result of inefficient post-transcriptional events such as 
5’ cleavage for trans-splicing or 3’ end formation for 
polyadenylation [13]. The steady state levels of hrcpb 
RNA correiate very well with the activities of the types 
A, B and C CPs that occur in different forms of L. 
mexicana: very high in amastigotes, low in metacycfic 
promastigotcs and undetectable in multiplicative pro- 
mastigotes [5]. 
nckr~o~vi~~~e/lr~~rs: This work was supported by the MRC and The 
Wellcomc Trust. Cohn Robertson is thanked for sharing unpublished 
results and critical reading orthe manuscript. David Laughland and 
John Laurie are thanked for technical assistnnce. 
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Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of Leishmania mexicana

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