Lymphopain, a Cytotoxic T and Natural Killer Cell-Associated Cysteine Proteinase J Brown1, E Matutes2, a Singleton3, C Price1, H Molgaard1, D Buttle3 and T Enver1

Leukemia (1998) 12, 1771–1781  1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu Lymphopain, a cytotoxic T and natural killer cell-associated cysteine proteinase J Brown1, E Matutes2, A Singleton3, C Price1, H Molgaard1, D Buttle3 and T Enver1

1Leukaemia Research Fund Centre at the Institute of Cancer Research, Chester Beatty Laboratories, London; 2Academic Haematology, Royal Marsden Hospital, London; 3Department of Human Metabolism and Clinical Biochemistry, University of Shefﬁeld Medical School, Shefﬁeld, UK

Through differential screening of established human leukaemia Results and discussion cell lines, we have identified and molecularly cloned lymphopain, a novel cysteine proteinase of the papain family. Lympho- pain exhibits a remarkably restricted cellular pattern of Molecular cloning of lymphopain expression, being predominantly expressed in cytotoxic T-lymphocytes and natural killer cells. The human lymphopain locus Human leukaemia cell lines have provided an invaluable maps to chromosome 11q13, encodes a polypeptide of 376 resource for the molecular analysis of the haemopoietic sys- amino acids and is conserved in the mouse. Both human and tem.6,7 We have used two such lines which have different murine forms appear more closely related to protozoan papain- haemopoietic lineage affiliations in a differential screen aimed like enzymes than to other mammalian members of the papain family. The cellular distribution of lymphopain expression, at identifying genes with lineage or stage-restricted patterns of together with the functional demonstration of lymphopain- gene expression within the haemopoietic system. One of associated proteinase activity in vitro, is suggestive of a role these, namely KG-1a is thought to represent a haemopoietic for lymphopain in immune cell-mediated, cell killing. progenitor cell with both lymphoid and myeloid potential.8–10 Keywords: lymphopain; cell killing; cysteine proteinase; LGL leu- The other, K562, is considered to represent a multimyeloid kaemia; T-lymphocytes progenitor with erythroid, megakaryocytic and monocytic potential.11 KG-1a mRNA was used to produce 32P-labelled single stranded first strand cDNA which was hybridised in sol- Introduction ution against an excess of K562 mRNA. Unhybridised cDNA was selected using hydroxyapatite chromatography and sub- The papain family of cysteine peptidases (family C1, see Ref. jected to another round of hybridisation. The resulting 1 for review) has representatives in bacteria, viruses, plants material was used to probe a KG-1a cDNA library prepared and animals. Although the major features of these enzymes in phage ␭. Two hundred positively hybridising plaques were are clearly conserved, the family can be further divided into picked and rescreened at lower density. Positives from this four distinct branches of a phylogenetic tree: the cathepsin B screen were replica plated and duplicate filters screened with branch, the plant branch, a branch containing enzymes from either KG-1a or K562 cDNA probes. Arrays of differential protozoa, and the cathepsin L branch.2 In mammals, some of clones were subsequently screened with cDNA probes the papain family enzymes, such as cathepsins B (EC derived from a panel of lymphoid and myeloid leukaemia cell 3.4.22.1), H (EC 3.4.22.16), L (EC 3.4.22.15), and dipeptidyl lines (KG-1, JM, KM3, U937 and ML1) and then used as pro- peptidase I (EC3.4.14.1) are found in many cell types as major bes on Northern blots of this same cell line panel. The three components of the lysosomal protein degradation system,3 clones which appeared to be substantially more highly though exceptionally, as in the case of bleomycin hydrolase, expressed in KG-1a and KG-1 than in the other cell lines they are cytosolic. Other mammalian members of the family, tested were sequenced. Two of these corresponded to genes such as cathepsin S (EC 3.4.22.27) and cathepsin K (EC which are known: ISGF-3, initially described as an interferon- 3.4.22.38) (also known as cathepsin O2), appear to have more dependent transcription factor12 and now known as STAT-1, specific functions in antigen processing,4 and bone resorp- and NK-4,13 a gene expressed in activated natural killer cells tion,5 respectively, and these functions are reflected in their and T cells. ISGF-3/STAT-1 and NK-4 are represented eight distribution. We now report on a new mammalian cysteine and 18 times, respectively, amongst the differential clones. peptidase of the papain family. This enzyme, which we have The third differential clone, designated A17, was represented named lymphopain, was cloned as part of a differential screen 14 times and did not appear in any of the sequence databases aimed at isolating molecules which exhibit lineage-specific examined and was therefore selected for further analysis. A17 patterns of expression within the haemopoietic system. Sur- was named lymphopain (Lpn) to reflect its KG-1/KG-1a speci- prisingly, lymphopain appears to be more closely related to ficity, its sequence homology to papain-family proteinases and the papain-like enzymes from protozoa than to mammalian its amidase activity (see below). papain-family members. The gene maps to 11q13 in humans and is conserved in the mouse. Lymphopain expression is largely restricted to the haemopoietic system and in particular to Tissue distribution of lymphopain expression the T cell compartment where it is associated with cytotoxic T lymphocytes (CTL), and natural killer cells. This cellular dis- The expression pattern of lymphopain was further examined tribution, together with the demonstration that lymphopain by Northern blot analysis of normal human tissue mRNAs. The encodes a functional enzyme, leads us to speculate that lym- lymphopain probe detected a 1.3 kb mRNA which is highly phopain may play a role in CTL/NK cell-mediated cell killing. expressed in spleen and peripheral blood leukocytes; low level expression is seen in the thymus and some other tissues (Figure 1a). No expression of lymphopain was observed in Correspondence: T Enver, Leukaemia Research Fund Centre at the Institute of Cancer Research, Chester Beatty Laboratories, Fulham either foetal liver or foetal spleen and little or no expression Road, London SW3 6JB, UK; Fax: 0171 352 3299 was seen in adult lung or liver samples (Figure 1b); both of Received 10 June 1998; accepted 7 July 1998 these tissues are rich in cysteine proteinases. No lymphopain Lymphopain: a lymphocytic cysteine proteinase J Brown et al 1772 a expression profile which was examined in more detail by subfractionating peripheral blood (Figure 2).

,0 ,m; I •::a Ill• E. fij: ';.:I to Expression of lymphopain in lymphocytes il:li, 0 ,s a !I. i::I! •• Two different peripheral blood samples were analysed and in both cases lymphopain expression was associated with the ILymphop n lymphocyte-containing fractions 1 and 4 which contain 25% and 95% lymphocytes, respectively. These results are consistent with analysis of lymphopain expression in haemopoietic GAPDH cell lines where no lymphopain expression was observed in K562 (CML in myeloid blast crisis), HL-60 (AML), ML-1 (AML), GI •·· n U937 (monocytic), or THP-1 (monocytic). Furthermore, the lymphopain signal observed in the lymphocyte fraction was substantially reduced by depletion of T-lymphocytes using erythroid cell rosetting (see Methods) and the human B cell lines that were tested (Nalm 6, KM3, REH, Daudi) were all b E negative for lymphopain mRNA (not shown). These data J - suggest a predominantly T-lymphocyte expression profile for lymphopain and peripheral T-lymphocytes were therefore subfractionated into CD4- and CD8-positive subsets using antibody-coated beads; the purity of these immunopurified populations was confirmed by FACS analysis (Figure 3a). Northern blot analysis of lymphopain expression in these samples is shown in Figure 3b. The results are not immedi- ately striking but suggest expression in both CD4- and CD8- positive cells, although expression in CD4-positive cells appears weaker. These data suggest that expression within T cells may be more complex than originally anticipated, a notion also supported by the low level expression seen in the thymus and the observation that lymphopain expression in T cell lines is variable (not shown). Expression in T cells and in

FRli_CIT ION 1 2 ! 4 1.31Kb 7IJi IIUi ill. ·1 ~ - Ill iH, 0 ;(I 0":N~ I) i O D 4 0 1GAPDH 1.3~ ;, 1..S U Qi ~. ;

Figure 1 Northern blot analysis of lymphopain expression in human tissues. (a) Lanes contain 2 ␮g of poly A+ RNA isolated from the tissues indicated (Clontech). The membrane was rehybridised with GAPDH and globin probes to control for sample loading and blood cell contamination of tissues respectively. (b) Human foetal spleen, foetal liver and embryonic skin samples contain 2.5 ␮g of total RNA. Adult lung and liver samples contain 0.6 ␮g of poly A+ RNA. The membrane was rehybridised with an actin probe to control for sample loading. (c) Lymphopain probing of human bone marrow (15 ␮g) and KG-1 (15 ␮g) samples; GAPDH probing provides a loading control. h: i f f J - Al:Jl111 - •~j . ' . - 2'.0:Kb expression was observed in embryonic skin (Figure 1b), foetal .... __._ ----~ skin, adult skin, keratinocytes or sebocytes (not shown). Simi- larly lymphopain expression was not detected in breast cancer Figure 2 Northern blot analysis of lymphopain expression in frac- cells (T47D), neuroblastoma (IMR32), hepatocellular carci- tionated peripheral blood from two normal individuals. Fraction 1, noma (HepG2), salivary gland carcinoma (HSG), undifferen- buffy coat; fraction 2, lymphocyte-depleted buffy coat lysed with NH4Cl to remove red blood cells; fraction 3, lymphocyte-depleted tiated embryonal carcinoma (T2 Cl3) or embryonal carcinoma cells induced to differentiate by retinoic acid treatment (not buffy coat without red cell depletion; fraction 4, mononuclear cells. The cellular composition of the different fractions was assessed by shown). High levels of lymphopain mRNA were however seen morphological analysis (see Methods). From left to right, lanes were in adult primary bone marrow cells (Figure 1c). Taken together loaded with approximately 4.0, 1.9, 2.7, 10, 3.7, 1.6, 3.8, 10, 10 ␮g these data are consistent with a primarily haemopoietic of total RNA and sample loading is controlled for by actin probing. Lymphopain: a lymphocytic cysteine proteinase J Brown et al 1773 • Figure 3 Analysis of lymphopain expression in the lymphocyte compartment. Peripheral T-lymphocytes from two normal individuals (samples 1 and 2) were purified using anti-CD4 or anti-CD8 coated beads. The purity of the enriched (post column +ve) populations is shown in the FACS plots in (a). Total RNA from these populations was analysed by Northern blotting with lymphopain and actin control j probes (b). RNA loadings from left to right in ␮g are approximately: 4, 6, 2.5, 6, 6, 8, 7.5, 3.3, 8.4, 2.4, (as judged by UV spectro- photometry). (c) Lymphopain expression in primary human leukaemia samples (RO, NG, EK, BN, HL, SM, EJ, MK, BY): the immunophenotypic and clinical description of these leukaemias is given in Table 1. Note strong expression in RO, NG, HL and SM. No expression was observed in EK, BN or EJ even on significantly longer exposures than those shown. Trace expression was detected in MK and BY; this may reflect some expression in B cells or cell type heterogeneity in the primary leukaemic samples. Also included is the analysis of RNAs from established human leukaemia cell lines, NKL (natural killer), Jur- i kat (pre-T lymphocytic), and KG-1, 2.6 ␮g of RNA is loaded in each lane.

particular the CTL/NK compartment, was further examined by analysis of a variety of primary leukaemia samples where T cell subsets are clonally expanded thereby facilitating molecular analyses.

Analysis of lymphopain expression in lymphocytic leukaemias

Blood mononuclear cells from nine patients with a lymphoid malignancy were analysed. All cases had Ͼ90% leukaemic -1.IICb cells. The diagnosis was based on clinical features, cell mor- phology, immunophenotype and, when required, cytogen- etics and/or analysis of the TCR chain gene configuration (see Table 1). Four of the T cell samples corresponded to large granular lymphocyte (LGL) leukaemia, a disease characterised -:1.11111 by the clonal proliferation of cytotoxic and/or natural killer cells. Of these large granular lymphocyte leukaemias, two had a cytotoxic phenotype whose cells expressed CD8 and the natural killer marker CD16 (SM, NG), one was CD4− CD8− and expressed only natural killer cell markers (RO) and another had a CD4+ phenotype with coexpression of the natural killer marker CD56 and thus might represent the expansion C of a very rare natural killer cell subset whose cells are CD4+. _,_ The other T cell samples analysed included a T cell prolymphocytic leukaemia (mature) and two T cell acute lymphoblastic leukaemias (immature), one was CD4+ whilst the other t-..- + was CD8 . The two cases with a B cell malignancy corresponded to an immature or common acute lymphoblastic leu- _..,. kaemia and to a mature B cell disease, a B cell chronic lymphocytic leukaemia. --- Strong expression of lymphopain was only observed in the large granular lymphocytic leukaemia samples (RO, NG, HL and SM). Expression was probably not simply a function of the maturity of these samples, as lymphopain expression was d not detected in a mature T cell prolymphocytic leukaemia sample (EJ). These data are consistent with the failure to i I • uncategorically assign lymphopain expression to either CD4- or CD8-positive T cell subsets: the large granular cell leukaemias which express lymphopain are heterogeneous with regard to CD4 and CD8 expression and the clearly CD4 single-positive (EK) and CD8 single-positive (BN) acute lymphoblastic leukaemias do not express lymphopain. This large -~ granular cell association of lymphopain expression is intriguing. This population of cells is associated with the natural killer cell compartment14 and all the lymphopain-positive leukaemias express at least one of the granular/killer cell mar- Lymphopain: a lymphocytic cysteine proteinase J Brown et al 1774 kers, CD16, CD56 or CD57. Furthermore, the KG-1/KG-1a Figure 4 Nucleotide sequence of the human lymphopain locus. cells from which lymphopain was cloned appear to express a The sequence is compiled from the analysis of cDNA and genomic number of natural killer cell associated molecules and this is clones. Coding regions are in upper case and flanking regions and reflected in our cloning of NK-4 and ISGF-3/STAT-1 in our intronic sequences are shown in lower case. Predicted amino acid differential screen. Finally the expression of lymphopain is translation of the coding regions is shown below the corresponding nucleotides. The probable final residue of the signal peptide and the seen in the natural killer cell line NKL (Figure 3C). On the first residue of the mature protein are underlined. The active site resi- basis of these data we speculate that lymphopain may play a dues are italicised and underlined. These sequence data are available role in cytotoxic T and natural killer cells, most likely in cell- from GenBank under accession numbers: AF013611, AF015954. mediated cell killing where both serine (granzyme) and cysteine (caspase/dipeptidyl peptidase I) proteinases have been shown to be involved. Mapping and evolutionary conservation of lymphopain

Structure of the human lymphopain locus The lymphopain gene was mapped to chromosome band 11q13 by FISH analysis (Figure 5a, see Methods). This Two strategies were taken to obtain a full length lymphopain chromosomal localisation was consistent with mapping data coding region: (1) KG-1 cDNA libraries were PCR-amplified obtained by PCR analysis of a panel of monochromosomal using internal primers from the lymphopain coding region human–rodent hybrids. Human-specific lymphopain signals together with primers to cloning vector sequences; (2) PCR were only seen in hybrid JICL4 which contains human chro- screening of a human P1 genomic library with lymphopain- mosome 11 and hybrid HORL1 which contains both human specific primers was undertaken. Both genomic and cDNA chromosome 15 and the long (q) arm of human chromosome clones were sequenced and the results of this analysis, 11 (Figure 5b and data not shown). In addition to the product together with the predicted amino acid sequence are corresponding to human lymphopain, the lymphopain primers presented in Figure 4. The human lymphopain gene is com- used detected additional bands in the hybrid panel; a band of prised of 10 exons distributed over a DNA interval of approxi- 1.4 kb in the human–mouse hybrids and a band of 1.3 kb in mately 4 kb and is predicted to encode a translation product the human–hamster hybrids suggesting that lymphopain-like of 376 amino acids. Analysis of this predicted amino acid sequences may be conserved in rodents (Figure 5b). This sequence identifies lymphopain as a member of the papain notion was further supported by Southern blot analysis of (C1) family of peptidases.1 As with almost all members of this murine genomic DNA using a human lymphopain cDNA family, its cDNA encodes a putative signal peptide and pro- clone as a probe (Figure 5c); this analysis also raises the possi- peptide, suggesting that it is a secreted protein, destined either bility that lymphopain-related sequences may also be for the extracellular milieu or the lysosomal/endosomal com- presented in the pig genome. The PCR product observed in partment. Transient transfection experiments in COS cells the mouse was sequenced and the remaining murine lympho- using a c-terminal epitope (myc) tagged Lpn cDNA have pain coding region was obtained using 5Ј and 3Ј RACE analy- yielded inconclusive results with regard to determining the sis of murine spleen mRNA. The sequence of these PCR pro- intracellular localisation of lymphopain (data not shown). ducts was confirmed by DNA sequencing of subsequently While some evidence for lysosomal compartmentalisation has isolated murine genomic clones (J Brown and T Enver, been obtained, the predominant pattern of staining is consist- unpublished). Human and mouse lymphopain coding ent with localisation to the endoplasmic reticulum. This may sequences are 76% identical at the nucleotide level. An align- reflect the inability of COS cells to appropriately ‘handle’ ment of human and mouse predicted amino acid sequences ectopically expressed lymphopain or may result from c- is shown in Figure 5d. The pre-pro-proteins display 69% terminal processing problems with the myc-tag. identity and 79% similarity; the predicted mature enzymes

Table 1 Immunophenotype of lymphoid leukaemias

Case CD2 CD3 CD4 CD5 CD7 CD8 CD16 CD56 CD57

Large granular lymphocyte leukaemia 1-SM ++−−+++−+ 2-NO + cyt+−++++ − 3-RO ++−−+−++− 4-HL ++++−−−++

T cell prolymphocytic leukaemia 5-EJ +++++−−−−

T cell acute lymphoblastic leukaemia (TdT+) 6-EK − cyt++++− 7-BN + cyt+− + +

B cell malignancies 8-MK SmIg+ (lambda), CD79b+, FMC7+, CD10−, CD2− 9-BY TdT+, CD19+, CD10+, CD22+, CD56−, CD2−

A marker was considered positive when over 90% of leukaemic cells expressed the antigen recognised by the monoclonal antibody. The level of T cell contamination in B cell diseases was 9% and 2% as assessed by the staining with CD2 and CD3. cyt, cytoplasmic positive but membrane negative. Lymphopain: a lymphocytic cysteine proteinase J Brown et al

ttgtgggttctaggggtccaccctccacccaccaagcctgtcttccccttcgtggtttctccagccccgccctcacaccgtcctcctcgatctgcgcggc 100 1775

ttcctgcctccatgccactccagactgcaccggcATGGCACTGACTGCCCACCCCTCCTGCCTCCTGGCCCTGTTGGTGGCAGGCCTAGCCCAAGGCATC 200 MALTAHPSCLLALLVAGLAQ~I

AGAGGCCCCCTTAGGGCCCAGgtaagtgctcccaaacccttacctatgccagtcccacgcctggggtcaccccttcagaaacagctggcctgtcattctc 300 RGPLRAQ atttttcagagggagttgctgaggct ggagagggggctgacgtgcccaagggcacatgggaagacagaaaaggctaactggc t gacccctagcccagtcc 400 ttgcacggctgggaactaaggtggggccccagcaatgattcctctgcccactgcccctctccacccttggttccttgccagGACCTAGGTCCCCAGCCGC S00 DLGPQPL

TAGAGCTGAAAGAGGCCTTCAAGTTGTTCCAGATCCAGTTCAACCGGAGTTACCTGAGCCCAGAAGgtatcacagggcacatacatctccagtccaagcc 600 ELKEAFKLFQIQFNRSYLSPEE cccact t t t t t t t t t t t gagacggagt t tea ct ct tgt tgaccaggctggagtgcaat ggt gt cat ct tggctcactgcaacctccacct cccgggt t ca 700 agcgattctcctgcttcagcctctgagtagctgggatt acaggcatgcgccaccacacttggctaacctttacatttttagtagagacggggtttgacca 800 tgt tggccaggc tggt ct tgaac tc ctgacatcaggtgatc cgcctgct t egg ct tee caaagggc tgggat tacaggtgtaagccacggagcccagcca 900 tcccgct t t gtaataga tgaagaaa tcgaagct cagggtgggaaacagg catggccaaggccact cagcaggac aggccagggctggggg tgtgt tee ct 1000 ggtttcccgaaggcatgagtgggtccaggatcattctttgctatggatctggagggcagtggcaggaggtggggtcctgccctagtcctacgtgatcccc 1100 tggctccacagccacttccctgcgattctaggcagtggacagggggccaccacctagtcatttgctcattcagcaaacacccaaggacacccattttgtc 1200 cccacat tggggac t tc t tct c tagccca t9at c taacccc tgcccc tgccctctcgctgtgctga tctctctagggggctga tctgtgt gtgcc t t t ta 13 00 gcccagactgcctgtgccttgtcatctcctctccctgccatgttccagctctcctcctgtggtcagaggtgtaaaggacgtggcctggcaggaagctggt 1400 ctccagccacacatcc t tgagt caccaccaccggacccct tgcctggc t t cccacctagggtggcc agagatggtcctctggctgcct ctggggaaaa cc 1500 caaccctcggtgggctgggtaaccacttcctgggcctgggtatctgcagtggggccttgtgggggctacaggaaaggagccagtgctcaactgggtgggt 1600 gtgggtggaagggtgcccagccagc ggc tact t cc tgcccc taagggc t tgggagtcagcctagga caac tc cc t t t tggtc cagAGCATGCTCACCGCC 170 0 H A H R L

TGGACATCTTTGCCCACAACCTGGCCCAGGCTCAGAGGCTGCAGGAGGAGGACTTGGGCACAGCTGAGTTTGGGGTGACTCCATTCAGTGACCTCACAGg 1800 DIFAHNLAQAQRLQEEDLGTAEFGVTPFSDLTE taccat taa ccc t tctggct cg taggtggtggt tgggaagcgatctccccagggact c tggtagctggacccag cggacct t caat t t t tact t cccagg 1900 gaaggaaat t cat t tee ctgaggac caggggct tgggtgctggtatcaaatcacccag tgcagggg cagaatct ctgcagtc ctgtgtcc taa tccagcc 2000 taggggc cagcgcag-cagctgcagggaagcacc cagcagggagcc cage c tctc tagg ct tggcaccct tg-cct t tagggag acagggt ct tgct c tg t t 2100 gtccaggctgcagtacaatagtgcaatcacagctcactcagcctccaccctcctagactcaaagcaattcccccgacccctcaagccttcttaagtagct 2200 ggggctacaagccgcacaaccacgc ccagctaa t t t tctac t tt tgtgg agatgggtc tcggactcctgacctcaagtgatc cacccacc t tggcctccc 2 3 00 aaagtgctgggattataggtgtgagccaccacgcccggcctatccttgcctttttgcccctcagggaattatgggaccaacagctggagtgggaga9gca 2400 gaacagggagaaggggt caaaatgg agacc tcagtggccc tgt ctgt tc cccagAGGAGGAGTTTGGCCAGCTCTATGGCTATCGGAGGGCAGCTGGAGG 2 S00 EEFGQLYGYRRAAGG

GGTCCCCAGCATGGGCAGAGAAATAAGGTCTGAAGAGCCAGAGGAGTCAGTACCTTTCAGCTGTGACTGGCGGAAGGTGGCCGGCGCCATCTCACCCATC 2600 VPSMGREIRSEEPEESYPFSCDWRKVAGAISPI

AAGGACCAGgtatctgccgctacccagctggctctaattcagctaagtggtgggagggagagggtcacggcccgaacacctagccccgccccaccctctc 2700 K D Q gccctccagAAAAACTGCAACTGCTGCTGGGCCATGGCAGCGGCAGGCAACATAGAGACCCTGTGGCGCATCAGTTTCTGGGATTTTGTGGACGTCTCCG 2800 K N C N C W A M A A A G N I E T L W R I S F W D F V D V S V

TGCATGgtagggttgggagagggtgcgcgtgtgacaggggagggaggcctaggggcctggtcacccacactgtcccttcttgcaccagAACTGCTGGACT 2900 H E L L D C

GTGGCCGCTGTGGGGATGGCTGCCACGGTGGCTTCGTCTGGGACGCCTTCATAACTGTCCTCAACAACAgtgagtggactgccccgccactctggacatc 3000 G R C G D G C H G G F V W D A F I T V L N N S tgcaggggg acagggtgggcaggagcggaacct cct ccct tgt ct tgct tatctgcagGCGGCCTGGCCAGTGAAAAGGACT ACCCCTTCCAGGGCAAAG 3100 GLASEKDYPFQGKV

TCAGAGCCCACAGGTGCCACCCCAAGAAGTACCAGAAGGTGGCCTGGATCCAGGACTI'CATCATGCTGCAGAACAACGAGCACAgtgcgggcagggcagg 3200 RAHRCHPKKYQKVAWIQDFIHLQNNEHR gacacgggcggatggcagggacagacaccggggcagaggcagacacgctgggctactgggctagaagacaagagggggtggggggagcgaaggagcgaga 3300 gacccacacacccacatgccaagggtctgatgatgttcctgtccccagGAATTGCGCAGTACCTGGCCACI'TATGGCCCCATCACCGTGACCATCAACAT 3400 IAQYLATYGPITVTINM

GAAGCCCCTTCAGgtgagatgggggagctgatggggaaggggcatacaggagacttggtcccacactcagcccctcggcccccaccccctgcagCTATAC 3500 KPLQ LY

CGGAAAGGTGTGATCAAGGCCACACCCACCACCTGTGACCCCCAGCTTGTGGACCACTCTGTCCTGCTGGTGGGTTTTGGCAGCGTCAAGTCAGAGGAGG 3 600 RKGVIKATPTTCDPQLVDHSVLLVGFGSVKSEEG

GGATATGGGCAGAGACAGTCTCATCGCAGTCTCAGccrCAGCCTCCACACCCCACCCCATACTGGATCCTGAAGAACTCCTGGGGGGCCCAATGGGGAGA 3700 I W A E T V S S Q S Q -P Q P P H P T P Y W I L K J1. S W G A Q W G E

GAAGgtgagtgtgatc tat tgggggagggggcaaggcagaacaggcctc t t cccacct tcccgcccc ta tgt cccctaacct cctagGGCTATTTCCGGC 3800 K GYFRL

TGCACCGAGGGAGCAATACCTGTGGCATCACCAAGTTCCCGCTCACTGCCCGTGTGCAGAAACCCGATATGAAGCCCCGAGTCTCCTGCCCTCCCTGAac 3900 HRGSNTCGITKFPLTARVQKPDMKPRVSCPP ccac ctggc cccctca9ctctgtc c tgt taggc caactgcc tcct tgccagccccacc cccaggt t t t tgccca t cctc ccaa t c tcaatacagcctg aa 4000 taaaccaagacaagacctctggct tgtgaaa Lymphopain: a lymphocytic cysteine proteinase J Brown et al

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C c.> 00 .... I I 0 1\)1\)

Figure 5 Chromosomal localisation and evolutionary conservation. (a) Mapping the human lymphopain locus by FISH (fluorescence in situ hybridisation). The X-chromosome control probe is pseudocoloured in red and the lymphopain probe in green. (b) Chromosome mapping of the human lymphopain locus by PCR analysis of human–rodent monochromosomal hybrids. Typical data from the analysis of the hybrid panel are shown. The rodent origin of the individual hybrids is indicated; Ha, hamster; Mo, mouse. PCR using human lymphopain-derived oligomers (fwd: 5Ј-TGAGTTTGGGGTGACTCCATTC-3Ј, rev: 5Ј-CAAAACCCACCAGCAGGACAGA-3Ј) amplifies a 1.8 kb product corresponding to the human lymphopain locus as well as 1.4 kb and 1.3 kb products in mouse- and hamster-derived hybrids, respectively. These products were Southern blotted using the 32P-labelled fwd primer as a probe (upper panel). The lower panel shows hybridisation of the same filter using a human lymphopain-specific oligomer (5Ј-GGTCTCTATGTTGCCTGCCGCT-3Ј), internal to the amplified product, as a probe. (c) Zoo-blot analysis. Genomic DNA from the sources indicated was restricted with EcoRI (E) or BamHI (B) and Southern blotted using a full length human lymphopain cDNA probe. Numbers on the right are in kilobase pairs and represent the migration of the ␭-HindIII molecular weight marker. For the exposure shown the filter was washed at a stringency of 1 × SSC at 50°C. (d) Multiple amino acid alignment of human and murine lymphopain with CP5 from Dictyostelium discoideum, papain from Carica papaya, cruzipain from Trypanosoma cruzii and human cathepsins B and L (numbering according to the papain sequence). The initial alignment was produced using the PILEUP package (UWGCG, Daresbury, UK) and edited manually using the crystal structure of papain as a point of reference. This alignment was scored using the AMAS software package (Barton, Oxford, UK); human and mouse lymphopain form one subgroup, CP5, papain, cruzipain, cathepsins L and B from another. The alignment was scored at a threshold setting of 6; positions boxed in red are identically conserved in all the sequences analysed. Those boxed in blue show identity within one subgroup only. Positions boxed in green show similarity in one or both of the subgroups. The probable final residue of the signal peptide and first residue of the mature forms are indicated by arrowheads labelled S and M, respectively. Two potential N-linked glycosylation sites in human and murine lymphopain are indicated by open purple circles. Propeptide residues normally conserved in the ERFNIN subgroup15 of family C1 peptidases1 are indicated by open yellow circles. The active site residues are marked by filled black circles. The normally invariant Gly 23 position is indicated by an open blue circle. The positions of the C-termini of mature cathepsin B46 and cruzipain,47 resulting from C-terminal processing, are shown by arrowheads labelled B and Z, respectively. The mRNA from human lymphopain encodes for a protein of 376 residues, five more than the murine enzyme. The native cruzipain sequence extends for an additional 118 amino acids beyond those shown in the alignment. Sequence information was obtained from the following sources: papain, SwissProt PAPAFCARPA; cruzipain, SwissProt CYSPFTRYCR; human cathepsin B; SwissProt CATBFHUMAN; human cathepsin L;5 CP5, EMBL L36205. The murine lymphopain sequence data are available from GenBank under accession number AF014941. Lymphopain: a lymphocytic cysteine proteinase J Brown et al 1777 d s ,, 0

H. Lyrnpl1opoin l,\.lyr11phop111n CPS LOYn N .t. Popoin Cru2 tp:aln S l H A F E T Cathopoln L D H SL E AO C,1t hcptln 8

H. Ly111phop:aln r,1.Lymphop,1in CP~ P:ip:iln Cruzlp,1ln C.ithcp&lnl C:11110111:lnB

0 •

H. Lymphop.iln M.lymphop11ln CP5 Papa ln Cruilpeln Calhep1ln L Cathep,1ne

H. Lymphopain r.l.Lymphopoin CPS Pop11 ln Cruzlpaln Cathcµ~l11 L Cothep, inB It tl V N ; • H. Lymphopoln M. Lymphopain CPS Pop11ln Cru11p11ln Cuthepslnl C0Lh up1ln B !lU!'!l>.131!..JLLIOI.ILt;__l__LJI.J!l-"liillJ!l:..IJ.L.O..l:..lla..L_ ..LlLlL!l:..IJ..J.JUIJl.lll..l!l>C..I.Jl.Ll1__ .!Ll.l...l'-t.L..MJ!"""- -"-"ild.LI!:llL"-'SIUJ""'------• H. Lymphop,tn 1,1. Lympho~11~; ~;µ;l'H-1,---'!l-J~'-'!&!Hi\¥ Pap11ln Cruzipain c11111•p1lnl C1thep1lnB ...... Z B

Figure 5 Continued also display 69% identity. This degree of identity is somewhat is provided by the novel insertion in the lymphopain sequence lower than normally observed for species variants within this between residues 168 and 169 of papain, on the opposite side family. However the degree of nucleotide similarity and the of the molecule to the active site. The only other enzymes Zoo blot analysis argue strongly that the murine cDNA we with an insertion in this position are found in the slime mould have cloned is indeed the homologue of human lymphopain. Dictyostelium discoideum, one of which, CP5, is aligned with lymphopain in Figure 5d. The function of the insertion in the slime mould enzymes is thought to be to provide site(s) for Structural features of human and murine lymphopain phosphoglycosylation of certain serine residues.18 The novel insertion, although present in the mouse sequence, does not There are a number of conserved residues in the propeptide demonstrate a high degree of identity with either the human (Figure 5d) that place lymphopain in the ERFNIN subgroup of enzyme (33%) or with the sequences from the slime mould the C1 peptidases,15 distinct from cathepsin B. We therefore enzymes. predict that the conformation and function of the propeptide All the cysteine residues in the sequence of mature papain of lymphopain will be similar to those of related enzymes, are conserved in lymphopain, suggesting that all three of the forming a globular domain, and a segment that runs through disulphide bonds have been retained, and that the gross con- the active site cleft in the opposite direction to that of a sub- formation of the enzymes is similar. Amino acids implicated strate, sterically hindering access of substrate.16,17 Lympho- in the active site chemistry of papain19 are completely con- pain will thus presumably be activated by removal of the pro- served in the sequence of human lymphopain. These include peptide. Berti and Storer2 have phylogenetically classified the Cys25 and His159 (papain numbering; Figure 5d) which make ERFNIN subgroup of family C1 enzymes into three lineages: up the catalytic ion pair, Asn175 which orients the imidazo- the plant lineage, a lineage consisting largely of protozoan lium ring of His159 by means of an H-bond and Trp177 which enzymes (Ddis1) and the cathepsin L lineage, which also con- shields this bond from the solvent, as well as Gln19 of the tains another mammalian enzyme, cathepsin S. Representa- oxyanion hole. The S2 binding pocket (in the accepted tives of each lineage are aligned with human and mouse lym- terminology20) is important in defining the specificity of this phopain in Figure 5d. Human lymphopain shows only low family of peptidases, which generally favour hydrophobic resi- levels of identity with the mature enzyme sequences of any dues at this position. With the exceptions of the conserved of these (29% with the plant enzyme papain, 32% with cruzi- Trp69 and Val157, the residues lining this pocket are substituted pain from the parasitic protozoan Trypanosoma cruzii, and in lymphopain (Tyr67 → Phe, Pro68 → Val, Val133 → Thr, 29% with human cathepsin L). Thus, lymphopain may rep- Ala160 → Ser, Phe207 → Leu), implying that the substrate speci- resent a novel lineage of the C1 peptidases, having diverged ficity of lymphopain may be different to that of papain. Gly23 due to a different function. Further strength to this argument is almost invariant among the C1 peptidases. Where this resi- Lymphopain: a lymphocytic cysteine proteinase J Brown et al 1778 due is substituted it leads to a profound change of specificity a due to steric hindrance to entry of the substrate into the S1 pocket.21,22 The substitution Gly23 → Cys in lymphopain may therefore be expected to lead to a greater degree of selectivity of peptide bond cleavage than is evident with papain.

Functional studies of lymphopain

Preliminary experiments were directed at determining the cysteine peptidase activities in lysates of Lpn-transfected and mock-transfected COS cells. Using Bz-Phe-Val-Arg-NHMec as substrate, these experiments demonstrated that the transfected cells had a higher specific activity (953 nM/min/106 cells) than mock-transfected cells (527 nM/min/106 cells) at pH 5.5 in the presence of 4 mM cysteine. Using the other substrates tested (see below) and a pH of 7.2 instead of 5.5, the difference in activity between the transfected and mock-transfected cells was not as great (results not shown). The enzymatic activity of Lpn was then examined using lymphopain-programmed rabbit reticulocyte lysates (Figure 6a). The human lymphopain mRNA cell-free translation product was screened for activity against Bz-Phe-Val-Arg-NHMec, which is cleaved by most members of the C1 peptidase family. The highest activity was b found at pH 5.5 (not shown), so these conditions were chosen for further study. The activities of human lymphopain cell-free G translation product and a control were determined against this ti substrate, in the presence and absence of cysteine and E64. ... The experiment was repeated with new cell-free translation •e products, and in both experiments the activity of the Lpn sample was higher than the control in the presence of cysteine. i E64 inhibited the activity found in the lymphopain sample, ,i but not that in the control sample. The results of one of the two experiments are shown in Figure 6b. These results are 0.1 consistent with the presence of a C1 family peptidase in the _, lymphopain sample, but not in the control, and we conclude I u that we were measuring the activity of human lymphopain. The propeptide of the C1 family peptidases acts as an inhibitor of the enzyme, and must be removed for the 0 expression of full activity. Conditions in vitro that lead to activation of some of these enzymes include incubation at acidic pH with or without pepsin.23,24 However preincubation of the Figure 6 (a) 35S-methionine-labelled coupled transcription trans- human Lpn cell-free translation product under these con- lation reactions of sense (lymphopain) and antisense (control) human ditions failed to generate increased activity (results not lymphopain containing pBluescript subclones were fractionated by shown). It is known that the proforms of some C1 family pep- SDS PAGE and autoradiographed. The lymphopain-specific product tidases can express limited activity against small substrates,23 is arrowed and numbers on the right are in kilodaltons. Parallel reac- and that the active site cysteine remains accessible to small tions were used for assaying enzymatic activity of cell-free translation organic molecules much as amidase substrates and alkylating products against an amidase substrate (b). The products of cell-free translation of lymphopain mRNA were assayed using Z-Phe-Val-Arg- reagents such as E64, despite the presence of the pro- ° 25,26 NHMec at pH 5.5 and 37 C, as described in the Materials and peptide. methods section. Continuous-rate assays were used to record the level of activity in the buffer, then following addition of 4 mM cysteine, and finally after inactivation of a papain-like enzymes with 10 ␮M E64. Perspective (see Refs 27–32). Granzymes have also been suggested as Clearly, further work is required to elucidate the mechanism playing a role in number of other lymphocyte-associated pro- of activation of Lpn and the full spectrum of its activity. How- cesses27 including extravasation and migration of mature T ever the in vitro demonstration of enzyme activity associated cells. Granzyme A has been shown to cleave several extra- with lymphopain, coupled to its restriction to the cellular matrix proteins in vitro and granzyme B inhibits cytotoxic/natural killer compartment, raises the possibility that adhesion, and consequently growth of tumour cell lines. it plays a role in cell killing. Our cloning of the murine homo- Granzyme B expression has also been documented in murine logue of lymphopain will facilitate testing of this possibility multipotential FDCP-mix A4 cells33 and in mobilised CD34- through creation of targeted mutations in transgenic mice. positive cells.34 Our own preliminary results (not shown) sug- Such an approach has proved invaluable in the dissection of gest that lymphopain is also expressed in both these cell types, the cellular functions of the granzymes, granule-associated possibly suggesting that some components of granzyme and serine proteases which have been implicated in cell killing lymphopain regulation or function may be shared. Lymphopain: a lymphocytic cysteine proteinase J Brown et al 1779 Experimental procedures Chromosomal localisation by FISH

Cloning and sequencing Genomic DNA from (PAC/P1/cosmid) clones were labelled with biotin-14-d-ATP (Gibco, Paisley, UK) by nick translation as described previously.42 Probes were hybridised to normal Differential hybridisation/screening analysis was performed human metaphase chromosomes at 42°C for 2 h and strin- essentially as described by Davis.35 Briefly 32P-labelled single- gency washed in 0.4 × SSC at 70°C for 2 min. Probe signal stranded first strand KG-1a cDNA was hybridised in solution was detected with FITC-conjugated avidin and amplified with against excess microsomal or total K562 mRNA (rot 1/2 = FITC-conjugated anti-avidin (Vector, Peterborough, UK). ෂ3 × 103). Unhybridised cDNA was selected using hydroxy- Chromosome bands were identified by DAPI staining. Probe apatite chromatography and subjected to another round of signals were scored in 20 metaphase spreads using a Zeiss hybridisation. This probe was used to screen KG-1a cDNA (Oberkochen, Germany) Axiophot microscope equipped with libraries prepared in ␭gt 10 by established procedures. epi-fluorescence and a triple band pass filter. Digital images Libraries were estimated to contain 1–3 × 105 independent were obtained with a high resolution CCD camera clones.35,36 5Ј and 3Ј RACE (rapid amplification of cDNA (Photometrics, Tucson, AZ, USA) and Smartcapture software ends) was performed using the Clontech Marathon cDNA (Digital Scientific, Cambridge, UK). amplification kit according to the manufacturer’s instructions (Clontech, Palo Alto, CA, USA). Genomic P1 clones were obtained through sequential PCR-based screening of clonal pools (Genome Systems, St Louis, MO, USA). DNA sequencing of KG-1a cDNA clones was performed using M13 sub- Haemopoietic cells clones and the chain termination method and analysed by denaturing PAGE and subsequent autoradiography. Direct sequencing of PCR-derived products and genomic clones, as Fractionation of peripheral blood: 40 ml of heparinised per- well as verification sequencing of DNA constructs, was perfor- ipheral blood from two normal volunteers was used for the med using cycle sequencing and an automated ABI (Foster study. 5 mls were centrifuged for 20 min at 800 g and the City, CA, USA) DNA sequencer. DNA sequences were ana- buffy coat was retained (fraction 1). The remaining 35 ml were lysed using software packages available at Seqnet, Daresbury diluted v/v in HBBS, layered on Lymphoprep (Nycomed, and Barton. Oslo, Norway) and centrifuged for 30 min at 600 g. A mononuclear fraction was obtained (fraction 4), the Lymphoprep was discarded and the cell pellet centrifuged again for 20 min at 200 r.p.m. which resulted in a lymphocyte-depleted buffy Isolation and blotting of cellular RNA and DNA coat. Half of the latter was treated with NH4Cl to lyse the red blood cells (fraction 2) and the other half resuspended in Hank’s (fraction 3). All four fractions were washed three times Total cellular RNA was isolated from tissues or cell lines using in Hank’s prior to the molecular studies. Cytospin prep- the guanidinium isothiocyanate procedure of Chirgwin et al,37 arations were made and stained with May–Gru¨nwald-Giemsa or the one-step acid guanidinium/phenol/chloroform to estimate the cellular components on each fraction. The cell method38 when dealing with smaller numbers of cells. Poly- composition in the four fractions is given in Figure 2. In order adenylated (poly (A)+) RNA was isolated as previously to obtain a T cell-depleted fraction, 30 × 106 normal blood described39 by one cycle of chromotography on oligo (dT)- mononuclear cells were resuspended in 10 ml of RPMI-1640 cellulose (Collaborative Research). Poly A+ or total cellular and foetal calf serum and incubated with 10 ml of 1% sheep RNA was prepared for electrophoresis by denaturation in 50% erythrocytes in RPMI-1640 medium for 10 min at 37°C. There- formamide, 2.2 M formaldehyde and 20 mM phosphate, pH after, they were centrifuged for 5 min at 600 g and incubated 7.6, and run through 1.1% agarose gels, essentially as for 3 h at 4°C. The cell pellet was carefully resuspended in described.40 Gels were transferred in 20 × SSC to charged RPMI-1640, layered on Lymphoprep and centrifuged for nylon membranes (Amersham, Little Chalfont, UK) and 30 min at 600 g producing an interface layer, which was hybridised with ␣-32P-dCTP-oligolabelled probes as depleted of cells forming rosettes with sheep erythrocytes (E- described41 except that the hybridisation buffer was sup- rosette negative cells or non-T cells) and a pellet containing plemented with 0.2% PVP, 0.2% Ficoll, 0.1% SDS and the E-rosette-positive fraction (T cells). The E-rosette negative 0.2 mg/ml sonicated salmon sperm DNA. After overnight fraction contained B cells and monocytes and was free of T hybridisation at 65°C, membranes were washed in 0.5– cell contamination as assessed by staining with CD2 and CD3 0.1 × SSC, 0.1% SDS for 1 h and autoradiographed or phos- whilst the E-rosette fraction was CD2+ (99%+). Lymphocytes phoimaged (Molecular Dynamics, Sunnyvale, CA, USA). For from two normal volunteers were enriched for CD4+ and + isolation of genomic DNA, cells were lysed in 100 mM Tris- CD8 subpopulations by using magnetic sorting with CD4 and HCl, pH 8.0, 50 mM NaCl, 12.5 mM EDTA, 0.2% SDS and CD8 microbeads, according to the manufacturer’s instruc- digested overnight at 37°C with 100 ␮g/ml proteinase K. DNA tions. The purity of the fractions was assessed by testing them was extracted with phenol, ethanol-precipitated and resus- with monoclonal antibodies against CD4 and CD8. Mono- pended in distilled H2O. DNA was limit-digested with restric- nuclear cells from patient blood was obtained with consent, tion enzymes according to the supplier’s instructions and elec- according to the methods described above. FACS analysis was trophoresed on 0.8% agarose 1 × TAE gels prior to transfer in carried out using standard procedures on a Becton Dickinson + 0.4 M NaOH on to Hybond N membranes (Amersham). (San Francisco, CA, USA) FACS analyser equipped with LYSIS Membranes were hybridised with ␣-32P-oligolabelled probes II software. Established haemopoietic cell lines were cultured as described for RNA blots or with ␥-32P-end-labelled oligo- in RPMI 1640, supplemented with 10% FCS and 4 mM gluta- ° mers. mine, at 37 C, in 5% CO2 in a highly humidified incubator. Lymphopain: a lymphocytic cysteine proteinase J Brown et al 1780 Enzymatic assays emia cells: globin gene expression and methylation. Mol Cell Biol 1988; 8: 4917–4926. 8 Koeffler H, Golde D. Acute myelogenous leukemia: a human cell For cell-free translation human lymphopain cDNA was sub- line responsive to colony-stimulating activity. Science 1978; 200: cloned downstream of the T3 RNA polymerase promoter in 1153–1155. pBluescript (Stratagene, La Jolla, CA, USA). This plasmid was 9 Koeffler H, Billing R, Lusis J, Sparkles R, Golde D. An undifferen- used to programme a coupled transcription-translation rabbit tiated variant derived from the human acute myelogenous leuke- reticulocyte lysate system (TNT, Promega Biotec, Madison, mia cell line (KG-1). Blood 1980; 56: 265–273. WI, USA) according to the manufacturer’s intructions. Sample 10 Furley A, Reeves B, Mizutani S, Altass L, Watt S, Jacob M, van reactions were monitored by labelling with 35S-methionine den Elsen P. Divergent molecular phenotypes of KG1 and KG1a myeloid cell lines. Blood 1986; 68: 1101–1107. (Amersham) and subsequent SDS PAGE analysis. Continuous- 11 Lozzio B, Lozzio C, Bamberger E, Feliu A. 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