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HUMAN MUTATION Mutation in Brief #714 (2004) Online

MUTATION IN BRIEF

Loss-of-function Mutations in Cathepsin C in Two Families With Papillon-Lefvre Syndrome are Associated With Deficiency of Serine Proteinases in PMNs
Susanne F. de Haar1,2, D. (Ineke) C. Jansen1, Ton Schoenmaker1, Hilde De Vree3, Vincent Everts1,4, and Wouter Beertsen 1*
Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), Universiteit van Amsterdam, Amsterdam, the Netherlands; 2Department of Cell Biology and Histology, Academic Medical Centre, Universiteit van Amsterdam, Amsterdam, the Netherlands; 3Department of Periodontology, Dental School, Universiteit Gent, Gent, Belgium; 4Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), Vrije Universiteit, Amsterdam, the Netherlands *Correspondence to: Wouter Beertsen, Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), Universiteit van Amsterdam, Louwesweg 1, 1066 EA Amsterdam, the Netherlands; Tel.: +31 20 5188300; Fax: +31 20 5188512; E-mail: w.beertsen@acta.nl
Communicated by Mark H. Paalman
1

Papillon-Lefvre syndrome (PLS) is a rare autosomal recessive disease that involves severe periodontitis and hyperkeratosis of the hand palms and foot soles. Recently it was found that PLS patients carry loss-of-function mutations in the gene encoding cathepsin C (CTSC). In the present study we have analyzed the CTSC gene in two unrelated families with PLS. In the first non-consanguineous family, mutation analysis revealed the previously reported c.815G>C/p.R272P mutation. The second consanguineous family displayed a c.1213C>A mutation which resulted in the novel mutation p.H405N and is the first mutation described in the active site of the enzyme. The PLS patients had, next to the absence of cathepsin C activity in polymorphonuclear leukocytes (PMNs), no activity of the three serine proteinases elastase, cathepsin G and proteinase 3. Serine proteinases are supposed to be important in both the innate and adaptive immune systems. Their absence in PLS patients could explain the inadequate defense to periodontal infection. 2004 Wiley-Liss, Inc.
KEY WORDS: Papillon-Lefevre syndrome; PLS; periodontitis; cathepsin C; CTSC; elastase; proteinase 3; cathepsin G; PMN

INTRODUCTION

Papillon-Lefvre Syndrome (PLS; MIM# 245000) was first described by M.M. Papillon and P. Lefvre in 1924 (Papillon and Lefvre, 1924). PLS involves severe prepubertal periodontitis and palmoplantar hyperkeratosis. The incidence of the disease is estimated at 1-4 per million (Gorlin et al., 1964). In PLS patients, the gingiva becomes severely inflamed upon eruption of the teeth. The inflammation progresses rapidly and results in the destruction of the tissues supporting the roots of the teeth, often leading to premature loss of the deciduous dentition. After a brief edentulous period the process resumes during the early stages of the permanent dentition. The hyperkeratosis involves dry and scaly hand palms and foot soles during the first years of life. The extent of hyperkeratotic plaques varies considerably between patients and often involves, besides the palmoplantar regions, the knees and elbows as Received 31 October 2003; accepted revised manuscript 19 February 2004.

2004 WILEY-LISS, INC. DOI: 10.1002/humu.9243

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well. It has been suggested that periods of severe hyperkeratosis coincide with the occurrence of acute periodontitis (Ullbro et al., 2003). Recently, mutations in the gene encoding cathepsin C (CTSC, MIM# 602365) have been reported in PLS patients (Hart et al., 1999; Toomes et al., 1999). The CTSC gene consists of 7 exons and is located on chromosome 11q14-q21 (Fischer et al., 1997; Laass et al., 1997). Cathepsin C (Dipeptidyl peptidase I (DPPI); EC 3.4.14.1) is a 200 kDa proteinase that belongs to the papain family of endopeptidases subfamily C1A. Cathepsin C activity is found in most tissues but particularly in cells of the immune system (McGuire et al., 1997). In a cathepsin C knockout mouse model cathepsin C was pointed out as the activator of the serine proteinases elastase, cathepsin G and proteinase 3 in PMNs (Adkison et al., 2002). PMN derived serine proteinases have been shown to play a crucial role in the defense against pathogens. Research using mouse models indicated that cathepsin G is required for the killing of Staphylococcus aureus (Reeves et al., 2002). Elastase knockout mice have difficulties in coping with infections of Escherichia coli, Candida albicans or Klebsiella pneumoniae (Frohm et al., 1999; MacIvor et al., 1999; Reeves et al., 2002). In vitro, cathepsin G and elastase are able to kill Actinobacillus actinomycetemcomitans and Capnocytophaga spp. Peptides derived from cathepsin G are capable of killing Staphylococcus aureus (Bangalore et al., 1990). Although proteinase 3 resembles elastase both structurally and functionally, its precise function remains unclear. In the present study we describe gene analysis of the CTSC gene in two families with PLS and we demonstrate that in the PLS patients the absence of cathepsin C activity coincides with absence of activity of the serine proteinases elastase, cathepsin G and proteinase 3.
MATERIALS AND METHODS Patients and sample isolation

The parents of family 1 were born in Belgium and no consanguinity of the parents was reported up to three generations back (De Vree et al., 2000). Two of their 4 daughters presented severe prepubertal periodontitis and mild palmoplantar hyperkeratosis (Figure 1). Other than the characteristic symptoms of PLS, the girls were healthy. Family 2 is of Pakistani origin and the parents are first cousins (Petit et al., 1993). The youngest two girls of four children have been diagnosed as suffering from PLS based on the presence of severe prepubertal periodontitis and extensive palmoplantar hyperkeratosis (Figure 1). Both patients have also suffered from additional medical problems including recurrent skin infections and a severe liver abscess. After informed written consent, peripheral blood was obtained from all patients and their parents by standard venipuncture. Genomic DNA was isolated from the blood samples using the Puregene kit (Gentra systems Inc, MI, USA) according to the manufacturers protocol. The control group for family 2 consisted of 50 ethnic matched healthy unrelated individuals of Pakistani origin.
Sequencing and mutation analysis

Each of the 7 exons and adjacent intron/exon boundaries of the CTSC gene were amplified by PCR using primers described by Toomes et al. (1999) (exon 2-exon 6) and by Lefevre et al. (2001) (exon 1 and exon 7). Samples of PCR product were purified (Qiaquick, Qiagen, Hilden, Germany) and subsequently analyzed by 2% agarose gel electrophoresis. Sequence reactions were performed with BigDye terminator mix (Applied Biosystems, Foster City, CA, USA) and samples were sequenced on an Applied Biosystems Sequencer 3100. Sequence data were automatically collected and analyzed by using the ABI Sequence Analysis software. The DNA sequences were compared with published CTSC sequences (GenBank U79415.1). Nucleotide numbering was referred to the ATG startcodon, using the A as +1.
Restriction enzyme analysis and allelic discrimination

Verification of the mutations was performed by restriction enzyme analysis of purified PCR products. The mutation in exon 6 of family 1 eradicated an AccI restriction site leaving the PCR fragment of exon 6 intact upon digestion (New England Biolabs, Beverly, MA, USA). The mutation in exon 7 of family 2 destroyed one of the restriction sites of NlaIII (New England Biolabs, Beverly, MA, USA) present in exon 7. Cleavage with NlaIII did not affect the 300-bp fragment of the PCR product of exon 7. All digestions were carried out according to the manufacturers protocols. Fragments were analyzed by 2% agarose gel electrophoresis. Screening for the SNP in

Mutations in CTSC 3

exon 6 of family 2 was performed by allelic discrimination (Assay-by-Design, Applied Biosystems, Foster City, CA, USA) on an ABI Prism 7000 and data were analyzed using the software supplied by the manufacturer.
Enzyme activity analysis and western blotting

Polymorphonuclear leukocytes (PMNs) were isolated from peripheral blood samples by a continuous percoll gradient and subsequently depleted of erythrocytes by lysis (Hamers et al., 1984). The PMNs were lysed and the samples were normalized for protein content which was determined using a BCA protein assay kit (Pierce, Rockford, Ill, USA). Cathepsin C activity was measured by the release of fluorescence upon the hydrolysis of the synthetic substrate Gly-Arg-MNA as previously described by McGuire et al. (1992). Elastase and cathepsin G activity were measured by the hydrolysis of respectively N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA and Nsuccinyl-Ala-Ala-Pro-Phe-pNA as described by Claesson et al. (1994) with minor modifications. Proteinase 3 activity was measured by the hydrolysis of N-t-but-Ala-Ala-Nva-thiobenzylester according to Kam et al. (1992). (Substrates from Sigma Chemicals Co, St. Louis, USA) For western blotting, samples of PMN lysates were separated by 12% SDS-polyacrylamide gel electrophoresis under reducing conditions. The proteins were blotted on nitrocellulose membranes and incubated with primary antibodies against elastase (1:100) (Calbiochem Corp., La Jolla, CA) cathepsin G (1:500) (Calbiochem Corp., La Jolla, CA) or proteinase 3 (1:500) (Neomarkers, Fremont, CA). The membranes were stained with appropriate peroxide labeled antibodies (1:2000) (DAKO, Glostrup, Denmark). Immune reactive bands were visualized by fluorescence, using ECL-Plus (Amersham, England, UK).

RESULTS AND DISCUSSION

In family 1 the previously reported mutation c.815G>C/p.R272P was found (Toomes et al., 1999). The two patients were proven to be homozygous for this mutation. Although the parents were not consanguineous, both were heterozygous for the c.815G>C mutation. The results of the restriction enzyme analysis were in line with the sequence data.

Family 1

Family 2

I II
N/A

I II

N/A

N/A

N/A

Figure 1. The pedigrees of family 1 and family 2. Males are denoted by squares and females by circles. The affected family members are indicated by filled symbols, the open symbols represent clinically unaffected individuals. A horizontal double line indicates a consanguineous marriage. N/A: no data available.

In family 2, an SNP and a mutation were identified in the CTSC gene. The SNP was found in exon 6 and involved the nucleotide change c.826A>G. The mutation in exon 7 involved a c.1213C>A and caused the p.H405N mutation. Sequence data indicated that the PLS patients were homozygous carriers of both mutations, whereas the parents were heterozygous. The presence of the SNP was confirmed by allelic discrimination whereas restriction enzyme analysis was applied for the mutation in exon 7. Screening of the 50 ethnic matched controls by similar allelic discrimination and restriction enzyme analysis for respectively the SNP and the mutation of family 2 showed that none of the controls carried either of these changes. The patients of family 1 and family 2 showed no cathepsin C activity, whereas their parents presented reduced levels of activity. Measurements of serine proteinase activities in PMN cell lysates revealed that the two patients of family 1 and patient II:2 of family 2 do not have elastase, cathepsin G or proteinase 3 activities (for patient II:1 of family 2 was no sample available for analysis of serine proteinases). Western blotting showed that the patients did

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not have an immune reactive band at the predicted heights for either of these serine proteinases. The samples of PMN cell lysates of the parents of both families contained all three serine proteinase activities and the corresponding proteins have been detected by Western blotting (Figure 2).

kDa 25

Family 1

Family 2 Proteinase 3 Cathepsin G Elastase

25

25

II:1

II:2

I:1

I:2

II:2

I:1

I:2

Control

Figure 2. Western blot analysis of PMN cell lysates of family 1 and family 2. Equal amounts of protein were separated by SDS-PAGE gel electrophoresis and blotted to nitrocellulose membranes. A similar sample of a healthy subject was used as a control. Upon incubation of the membranes with antibodies against proteinase 3 and cathepsin G the parents of family 1 and family 2 displayed bands at respectively 27 kDa and 28 kDa. Incubation of membranes with elastase antibodies resulted in bands at 31 kDa for the parents, but at 28 kDa for the PLS patients.

The p.R272P mutation found in family 1 is located in a highly conserved region in CTSC that encodes part of the second turn of the central helix from the c-terminus of the cathepsin C (Figure 3A). The R272 is a single chain residue and is different in shape and charge compared to the ring structure of the mutated P272. Consequently the p.R272P mutation of family 1 is predicted to result in a distortion of the 3-D structure and a dysfunctional enzyme (Turk et al., 2001). The p.H405N mutation of family 2 directly affects one of the active sites of the enzyme. H405 is located in a region of 16 amino acids, which is highly conserved in the evolutionary track of cathepsin C (Figure 3A). Together with the C258 and the N427, it forms the catalytic triad, which is located just above the oxy-anion hole formed by the amides of Q252 side chain and C258 main chain (Turk et al., 2001). The enzymatic action of cathepsin C involves a two-step acylation-deacylation reaction. In the initial phase H405 accepts a proton from C258 and is then stabilized by N427 (Stryer, 1988). This shift in charge enables C258 to attack the substrate. Finally, after hydrolysis of the peptide bond, H405 returns a proton to C258. The importance of the catalytic triad is emphasized upon alignment of the 11 structurally related enzymes of the papain peptidase subfamily C1A (Figure 3B). The active sites of each of these enzymes consist of identical amino acids as in cathepsin C. Since the H405 is crucial for the enzymatic action of cathepsin C, it is likely that the p.H405N mutation caused the absence of cathepsin C activity in the patients of family 2. The majority of the 41 changes described up to now in the CTSC gene are missense mutations, which give rise to folding difficulties, prevent correct targeting, abolish binding sites for substrates and co-factors, or might lead to other yet unknown problems (Selvaraju et al., 2003). The remaining changes include nonsense mutations, insertions, deletions and a splice variant that usually result in a truncated form of the protein. As far as the hyperkeratosis is concerned, Hart et al. (2000) studied whether the type of mutation (deletion, insertion, substitution) could be associated with the presence or absence of transgression of the dermatological lesions. However, such a correlation was not found. A similar result was found recently by Selvaraju et al. (2003). In the present report, both patients of family 1 displayed a mild hyperkeratosis. In contrast, the patients of family 2 did not only show severe hyperkeratosis but also suffered from recurrent infections and from a severe liver abscess. Whether the severity of the dermatological lesions and the occurrence of other medical problems in these families could be related to the sort of defect in the enzyme (a distortion of the 3-D structure versus a defective active site) remains to be elucidated.

Mutations in CTSC 5

A.
human: mouse: rat: dog: schjp: schma:

Family 1: R272P (exon 6)

Family 2: H405N (exon 7)

?
MLEARIRILTNN MLEARIRILTNN MLEARIRILTNN MLEARIRILTNN AIEARIRLASRF ALEARIRLVSNF

?
FNPFELTNHAVLLVGY FNPFELTNHAVLLVGY FNPFELTNHAVLLVGY FNPFELTNHAVLLVGY FNPFELTNHAVLLVGY FNPFELTNHAVLLVGY

B.
cathepsin cathepsin cathepsin cathepsin cathepsin cathepsin cathepsin cathepsin cathepsin cathepsin cathepsin papain: C: K: B: H: L: X: S: O: W: F: V: C258 CGSCY CGSCW CGSCW CGSCW CGSCW CGSCW CGACW CGCCW CNCCW CGSCW CGSCW CGSCW H405 LTNHAVLL NLNHAVLA MGGHAIRI KVNHAVLA DMDHGVLV NLNHAVLA NVNHGVLV EANHAVLI LVDHSVLL LIDHAVLL NLDHGVLV KVDHAVAA N427 VKNSWG IKNSWG VANSWN VKNSWG VKNSWG VRNSWG VKNSWG VRNSWG LKNSWG IKNSWG VKNSWG IKNSWG

Figure 3. Alignment of highly conserved amino acid regions of CTSC and other homologous proteins. Alignment was performed by the use of Seqweb software. A) Alignment of the highly conserved amino acids flanking the mutations found in CTSC gene in the families described in this study with homologous regions of other known CTSC gene sequences: mouse (Mouse CTSC, U74683), rat (Rat CTSC, D90404), dog (Dog CTSC, AF060171), schjp (Schistoma japonicum, U77932), and schma (Schistoma manson, Z32531). The affected amino acids found in the PLS patients are indicated in bold and denoted by arrowheads. B) The alignment of active site residues and flanking regions of the papain peptidase subfamily C1A (adapted from Dickinson, D.P., 2002). The active site residues are indicated in bold on a gray background.

Periodontitis is a widespread disease in humans and is often associated with the presence of virulent gramnegative bacteria, such as Actinobacillus actinomycetemcomitans (A.a.) and Porphyromonas gingivalis (P.g.) (reviewed by Eley and Cox, 2003). The fact that in many healthy individuals the presence of A.a. and P.g. is not associated with periodontitis suggests that there are also other factors involved in the pathogenesis of the disease. The first line of defense against invading pathogens is formed by PMNs. It is well known that diseases affecting the functioning of PMNs often cause an increased susceptibility to infections, including periodontitis (Deas et al., 2003) We demonstrated that PLS patients of both families, irrespective of their mutation in the CTSC gene, had no elastase, cathepsin G and proteinase 3 activities in their PMNs. This finding suggests that in humans as well as in mice a similar activation of serine proteinases by cathepsin C occurs. In none of the PMN samples of the PLS patients immune reactive bands for the serine proteinases were found at the predicted heights. The finding that incubation of cell lysates of PMN patients with purified cathepsin C did not yield serine proteinase activity (data not shown) and that bone marrow of cathepsin C knockout mice show markedly reduced levels of immune reactive cathepsin G (Adkison et al., 2002) suggests that synthesis of the protein is decreased or that the protein is more susceptible to degradation. Incubation of the blots with elastase antibodies resulted for the three patients studied in a thick, but clearly lower molecular weight band, possibly the result of fragmentation. More importantly, since serine proteinases and serine proteinase-derived peptides are supposed to play an important role in the elimination of bacteria mediated by PMNs, the absence of protein and protein activity of serine proteinases found in the PLS patients could explain their predisposition to periodontal infections.

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ACKNOWLEDGMENTS

We would like to thank Prof. P. M. Frossard (Dept. of Biological and Biomedical Sciences, Fac. of Health Sciences, The Aga Khan University, Pakistan) for providing us the Pakistani DNA control group and A.J.M. Hoogeboom (Erasmus University, Rotterdam, the Netherlands) for her assistance with collection of the samples.
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Mutations in CTSC 7

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