MUTATION IN BRIEF

HUMAN MUTATION Mutation in Brief #434 (2001) O nline

© 2001 WILEY-LISS, INC.

Received 28 February 2001; revised manuscript accepted 4 June 2001.

Expression and Analysis of CLN2 Variants in CHOCells: Q100R Represents a Polymorphism, andG389E and R447H Represent Loss-of-FunctionMutationsLi Lin and Peter Lobel

Center for Advanced Biotechnology and Medicine, Department of Pharmacology, Robert Wood Johnson MedicalSchool-University of Medicine and Dentistry of New Jersey

*Correspondence to: Peter Lobel, 679 Hoes Lane, Piscataway, NJ 08854, USAContract grant sponsor: National Institute of Health; Contract grant number: NS37918

Communicated by Leena Peltonen

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal hereditary childhood disease. Thegene underlying LINCL, CLN2, encodes a lysosomal enzyme, tripeptidyl peptidase I (TPP-I),deficiency in which leads to lysosomal accumulation of autofluorescent materials accompanied bysevere neuronal atrophy. Mutational analysis was conducted to characterize different CLN2alleles. Two probands of Romany origin were found to be homozygous for an allele that encoded aprotein with two changes, designated Q100R+G389E CLN2. To distinguish potentialpolymorphisms from mutations, a recombinant expression system was used to investigateindividual constructs. Elevated levels of TPP-I activity in CHO cells expressing Q100R CLN2and background activity in CHO cells expressing G389E CLN2 clearly defines G389E as apathogenic mutation and indicates that Q100R is a polymorphism. Association of the R447Hmutation with a delayed onset form of LINCL in two separate families raised the question ofwhether R447H CLN2 retains residual activity. However, CHO cells expressing R447H CLN2had TPP-I activity comparable to that of neo transfected cells, indicating that any residual activitywas below the level of detection in this experimental system. © 2001 Wiley-Liss, Inc.

KEY WORDS: late infantile neuronal ceroid lipofuscinosis; LINCL; CLN2; TPP-I; loss-of-function; residualactivity; Romany.

INTRODUCTION

Late infantile neuronal ceroid lipofuscinosis (LINCL, MIM# 204500) is a fatal hereditary neurodegenerativedisease that affects children. This disease is caused by mutations in the CLN2 gene that encodes the lysosomalenzyme tripeptidyl peptidase I (TPP-I) (Sleat, et al., 1997, Vines and Warburton, 1999). Deficiency of TPP-I activityresults in accumulation of undigested protein substrate in lysosomes and leads to progressive loss of vision, motorand mental regression, and early death (Mole, 1998). No effective treatment is currently available.

To date, more than 30 mutations in CLN2 have been identified to be associated with LINCL(http://www.ucl.ac.uk/ncl/CLN2.html). In most cases, these mutations can be predicted to cause complete loss of

2 Lin and Lobel

function of the protein (e.g., nonsense mutations, splice-junction mutations and missense mutations that may resultin disulfide bond mispairing or elimination of key active site residues) (Sleat, et al., 1999, Lin, et al., 2001). However,in several cases, the effects of some changes are not clear and warrant further investigation.

Two unrelated patients of Romany ethnicity were found to be homozygous for an allele containing two changesthat were 2167 nucleotides apart and were not found in any other patients or unaffected controls. Both changesaffect the coding sequence, resulting in production of a Q100R+G389E variant protein (Sleat, et al., 1999). It is notimmediately obvious how these changes are related to LINCL, but there are four possibilities: 1) both could be loss-of-function mutations and either could cause LINCL; 2) each could be impaired-function mutations with bothrequired together to cause LINCL; 3) one could be a loss-of-function mutation and the other a polymorphism; or 4)there is a formal possibility that both could be polymorphisms linked to an as yet unidentified loss-of-functionmutation. In the one pedigree analyzed, both parents were heterozygous for both changes, shedding little light onthese possibilities. A clear distinction between polymorphisms and mutations is necessary for DNA baseddiagnosis, prenatal testing, carrier detection, prediction of clinical outcome, and genetic counseling.

Another CLN2 allele that bears further functional investigation is the R447H mutation. Most CLN2 mutationstypically result in onset of symptoms between ages 2-4 and death between ages 6-15. In contrast, the R447Hmutation is associated with delayed onset (ages 7-8) and protracted disease (lifespan 20-40 years) (Sleat, et al.,1999). Of the two separate families (4 patients) previously characterized, the R447H mutation is compoundheterozygous for either a R208X nonsense mutation or a IVS5-1G>C splice-junction mutation, both of which arepredicted to terminate the CLN2 protein upstream of the active site and thus are null mutations (Lin, et al., 2001). Itis not apparent why the R447H mutation is associated with a protracted phenotype, but in many lysosomal storagedisorders, residual activities are associated with milder phenotypes or delayed onset and protracted disease. It ispossible that R447H is such a mutation. However, at the time of the initial mutation survey, no activity wasdetected in postmortem brain specimens using the pepstatin-insensitive protease assay (Sleat, et al., 1999).Considering the lability of CLN2p (Sohar, et al., 1999) coupled with uncontrolled sample preservation in autopsyspecimens, and the availability of more sensitive methods to detect CLN2p/TPP-I activity (Vines and Warburton,1998, Sohar, et al., 2000), we were prompted to re-examine this question.

In order to distinguish polymorphisms and mutations, and to test if the R447H CLN2p carries residual enzymeactivity, recombinant human CLN2 cDNA variants were expressed in CHO cells and analyzed with respect toenzyme activity.

MATERIALS AND METHODS

PCR-based mutagenesis was used to introduce the Q100R (CAA to CGA), G389E (GGA to GAA) and R447H(CGT to CAT) changes into the human CLN2 cDNA sequence. PCR-amplified DNA was subcloned into theexpression vector pSFFV-neo (Fuhlbrigge, et al., 1988) and the entire coding region sequenced to confirm thepresence of the desired mutations and absence of unwanted changes. DNA was introduced into CHO cells usinglipofectamine (GIBCO) and resistant clones selected using G418. For activity assays and Western blotting, cellswere seeded into 6-well plates and cultured in DME/F12, 10% fetal bovine serum until monolayers reachedconfluence. Cells were washed and cultured in 1 ml serum-free medium for 48 hours, washed with phosphatebuffered saline, and lysed with a solution (0.5 ml) of 1% Nonidet P40, 150mM sodium chloride, 10 mM Tris pH7.5.Samples were clarified by centrifugation at 12,000 × g for 5 min (media) or 20 min (lysates) and analyzed for TPP-Iactivity measuring hydrolysis of the Ala-Ala-Phe 7-amido-4-methylcoumarin substrate using the end-point assay(Sohar, et al., 2000) after preactivation at pH 3.5 for 30 min at 37°C. Clarified lysates contained between 1.2 to 1.5mg/ml protein as determined by the Lowry assay (Lowry, et al., 1951). Samples (lysates, 8 µg/lane; media, 15.6µl/lane) were fractionated by SDS-PAGE on a 10% NuPAGE Bis-Tris gel (Invitrogen). CLN2p was visualized byWestern blotting using a rabbit antiserum prepared against hexahistidine tagged CLN2p (antiserum R72-5) andenhanced chemiluminescence (Renaissance, NEN).

RESULTS AND DISCUSSION

The Q100R, G389E and R447H mutations were individually introduced into the CLN2 cDNA and subcloned intothe expression vector pSFFV-neo. Stable transfected CHO cells were subcloned and examined for expression ofCLN2p and clonal homogeneity by indirect immunofluorescence (data not shown). Elevated levels of CLN2p were

Mutational analysis of CLN2 3

detected by western blotting analysis on select clones (Figure 1). When conditioned media and cell lysates wereassayed for TPP-I activity, cells transfected with the wild type CLN2 expression vector had increased levels of TPP-I activity (Figure 2). No significant difference in TPP-I activity was observed when comparing cells transfected withQ100R and wild type CLN2 constructs. In contrast, the TPP-I activity of cells transfected with G389E CLN2 wasessentially indistinguishable from that of neo-transfected control cells (Figure 2). These results unequivocallyidentify G389E as a pathogenic mutation that causes loss of CLN2p function, which is consistent with that theconservation of G389 among prokaryotic and eukaryotic CLN2-related proteases (Sleat, et al., 1999). These resultsalso indicate that Q100R is a polymorphism that is closely linked to G389E in the affected Romany patients.

FIGURE 1. Western blotting analysis and TPP-I activity assay on representative CHO cell clones. Cells and mediafrom CHO clones transfected with pSV2neo control, wild type, Q100R, G389E, and R447H mutant human CLN2 wereanalyzed for CLN2p expression by western blotting. Bands migrating at the positions of proform and mature CLN2pare indicated. Other bands represent non-specific binding of the antibody. TPP-I activity was also measured afterincubating the samples at pH 3.5 to convert proenzyme to the active form and was normalized to total cellularprotein.

FIGURE 2. TPP-I activity comparison between CLN2 mutants. Different CHO cell clonesstably transfected with wild type, Q100R, G389E, or R447H mutant human CLN2 or thepSV2neo control plasmid were analyzed for TPP-I activity in both cell lysates and culturemedia. Horizontal lines represent mean values for a given construct.

4 Lin and Lobel

The lack of a detectable increase in TPP-I activity in CHO cells transfected with R447H CLN2 (Figures 1 & 2)indicates that R447H had a severe effect on the function of the CLN2p. However, at present, we cannot exclude thepossibility that a low level of residual activity is masked by the endogenous TPP-I activity in CHO cells (e.g., signal-to-noise considerations). Another possibility is that the R447H CLN2p has no activity and a linked, as yetunidentified genetic modifier is responsible for the variant phenotype in these patients. If so, identification ofgenetic suppressors that can delay disease onset and progression may provide insights into alternative therapeuticstrategies. Further studies of the properties of the R447H mutant CLN2p are required to distinguish between thesepossibilities.

In terms of structure-function relationships, it is of interest that constructs that resulted in elevated levels ofTPP-I activity (wild type and Q100R) also generated significantly increased levels of intracellular mature CLN2p andsecreted proform CLN2p, whereas G389E and R447H did not. These findings suggest that in addition to possibledirect effects on TPP-I activity, the G389E and R447H mutations may adversely affect CLN2p structure and stability,reducing the steady state levels of the protein within lysosomes.

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