A novel mutation in PNLIP causes pancreatic triglyceride lipase deficiency through protein misfolding

A no el mu a ion in PNLIP causes panc ea ic iglyce ide lipase de iciency h ough p o ein mis olding And ás Szabó†,1, Xunjun Xiao*,1, Ma ga e Haughney*, Aylssa Spec o *,Miklós Sahin- Tó h†, and Ma k E. Lowe2,* *Depa men o Pedia ics, Child en’s Hospi al o Pi sbu gh, Uni e si y o Pi sbu gh Medical Cen e , Pi sbu gh, PA. †Depa men o Molecula and Cell Biology, Hen y M. Goldman School o Den al Medicine, Bos on Uni e si y, Bos on, MA. 1These au ho s con ibu ed equally o he manusc ip . 2To whom co espondence should be add essed. Running i le: p.T221M PNLIP causes p o ein mis olding Co esponding Au ho : Ma k E. Lowe, MD, PhD Child en’s Hospi al o Pi sbu gh o UPMC Di ision o Pedia ic Gas oen e ology, Hepa ology and Nu i ion 4401 Penn A e Pi sbu gh, PA 15224 Tel: 412-692-5412 Fax: 412-692-8906 [email p o ec ed] Abb e ia ions: BiP, immunoglobulin binding p o ein; ER, endoplasmic e iculum; GRP94, glucose- egula ed p o ein-94; IRE1, inosi ol- equi ing enzyme 1; PNLIP, panc ea ic iglyce ide lipase; PNLIPRP2, panc ea ic lipase ela ed p o ein 2; UPR, un olded p o ein esponse; XBP1, X-box binding p o ein-1 ABSTRACT Congeni al panc ea ic iglyce ide lipase (PNLIP) de iciency is a a e diso de wi h unce ain gene ic backg ound as mos cases we e desc ibed be o e gene sequencing was eadily a ailable. Recen ly, wo b o he s wi h PNLIP de iciency we e ound o ca y a homozygous missense mu a ion, c.662C>T (p.T221M) in he PNLIP gene (J. Lipid Res. 2014. 55:307-312). Molecula modeling sugges ed he subs i u ion would change he o ien a ion o esidues in he ca aly ic si e and dis up he unc ion o p.T221M PNLIP. To es he e ec o he p.T221M mu a ion on PNLIP unc ion, we exp essed wild- ype and p.T221M PNLIP in human emb yonic kidney (HEK) 293A cells and dexame hasone-di e en ia ed AR42J a acina cells. In bo h cellula models, wild- ype PNLIP was sec e ed in o he condi ioned medium whe e i was eadily de ec able by p o ein s aining, immunoblo o lipase ac i i y assays. In con as , mu an p.T221M was no sec e ed in o he medium, bu i was p esen in cell lysa es whe e i accumula ed in he insoluble ac ion. In acellula e en ion o mu an p.T221M esul ed in endoplasmic e iculum (ER) s ess as measu ed by ele a ed XBP1 splicing and inc eased le els o ER chape ones. Ou esul s demons a e ha he p esence o me hionine a posi ion 221 in he PNLIP p o ein sequence causes mis olding and agg ega ion o he p.T221M mu an inside he cell. The consequen loss o enzyme sec e ion adequa ely explains he clinical pheno ype o PNLIP de iciency epo ed o homozygous ca ie s o p.T221M. Fu he mo e, he abili y o mu an p.T221M o induce ER s ess sugges s ha his o m o PNLIP de iciency migh cause acina cell damage as well. Keywo ds: Lipase, a diges ion, p o ein mis olding, endoplasmic e iculum s ess esponse 2 1. In oduc ion Al hough congeni al panc ea ic lipase iglyce ide (PNLIP) de iciency is discussed in mos ex books o pedia ic gas oen e ology, he e a e only a ew epo s o pa ien s wi h e idence o his a e diso de (1-10). In each case, he PNLIP de iciency was demons a ed by enzyma ic assay o panc ea ic sec e ions. In no case, was a mu a ion in he p o ein o gene epo ed. A ecen e o o iden i y mu a ions in he PNLIP gene o ou pa ien s wi h absen lipase ac i i y in hei panc ea ic luid ailed o ind any nonsense o missense mu a ions ha could explain he de iciency (11). Thus, i has ne e been clea i he absence o PNLIP ac i i y in he panc ea ic sec e ion o he epo ed pa ien s esul ed om a gene mu a ion o ano he eason such as he p esence o a lipase inhibi o o echnical p oblems wi h he sample collec ion o wi h he lipase assay. Recen ly, Beha e . al. (2014) epo ed wo b o he s om a consanguineous ma iage who had clinical PNLIP de iciency wi h s ea o hea and a no el homozygous missense mu a ion in he PNLIP gene (12). The he e ozygous ca ie pa en s we e clinically una ec ed. A single base subs i u ion in exon 6 (c.662C>T) changed he amino acid a posi ion 221 (p.T221M). Th 221 is conse ed in all known PNLIP sequences. I is loca ed in he β9 loop, which con ibu es o he ac i e si e o PNLIP (13). In he c ys al s uc u e o human PNLIP, Th 221 o ms a hyd ogen bond wi h Asp193, a esidue in he Se -His-Asp ca aly ic iad (14, 15). Molecula modeling sugges s he subs i u ion o Th 221 wi h he la ge amino acid, me hionine, des abilizes he ac i e si e o PNLIP (12). The au ho s specula ed ha he b o he s’ s ea o hea esul ed om dec eased ac i i y o p.T221M PNLIP. To es he hypo hesis ha he p.T221M mu a ion al e s PNLIP unc ion, we exp essed Th 221 PNLIP and Me 221 PNLIP in HEK 293A and AR42J cells. We hen de e mined he e ec o he p.T221M mu a ion on PNLIP exp ession, sec e ion and ac i i y. Ou esul s demons a e ha he p esence o me hionine a posi ion 221 in he p o ein sequence causes 3 mis olding o p.T221M PNLIP. The mis olded lipase accumula es in he cell, is inac i e and is no sec e ed in o he medium. 4 2. Ma e ials and me hods 2.1 Nomencla u e Nucleo ide numbe ing e lec s coding DNA numbe ing wi h +1 co esponding o he A o he ATG ansla ion ini ia ion codon in PNLIP. Amino acid esidues a e numbe ed s a ing wi h he ini ia o me hionine o he p ima y ansla ion p oduc o human PNLIP. 2.2 Exp ession Plasmids, Mu agenesis and Adeno i us The cDNA encoding human PNLIP was ampli ied by PCR using ou p e iously ob ained ull leng h human PNLIP cDNA clone as empla e and subsequen ly subcloned in o he mammalian p o ein exp ession ec o pcDNA3 (In i ogen, Ca lsbad, CA) (16). The c.662C>T (p.T221M) mu a ion was in oduced by o e lap ex ension PCR. The sequence o all plasmid DNA cons uc s was e i ied by dideoxynucleo ide sequencing. Recombinan adeno i us ca ying a His- agged o m o wild- ype PNLIP o mu an p.T221M was gene a ed by Vi aques , Inc. (No h Libe y, IA). Con ol adeno i us was pu chased om Vi aques . 2.3 Cul u e and T ans ec ion o Mammalian Cells HEK 293A cells we e pu chased om ATCC (Manassas, VA) and main ained in DMEM supplemen ed wi h 10% e al bo ine se um, 1% ol/ ol uni s/ml penicillin and s ep omycin (#15140-122 GIBCO, Li e Technologies, G and Island, NY) a 37° C in a 5% CO2-humidi ied incuba o . Six een hou s p io o ans ec ion, cells (2 ml/well o 6-well pla es and 10 ml/dish o 10-cm cul u e dishes) we e seeded a 75% con luence. T ans ec ions we e ca ied ou using FuGENE 6 (Roche Diagnos ics, Indianapolis, IN). B ie ly, in 100 μl o Op i-MEM I educed se um medium (In i ogen), 5 μl o FuGENE 6 was mixed wi h 1.65 μg o plasmid DNA (pcDNA3, pcDNA3/PNLIP, o pcDNA3/ PNLIP p.T221M). The mix u e was hen added o he cells in each well o 6-well pla es. Fo RNA ex ac ion, a mix u e o 25 μl o FuGENE 6 and 10 5 μg o each plasmid DNA in 500 μl o Op i-MEM I educed se um medium was added o each 10-cm dish. AR42J a panc ea ic acina cells (ATCC #CRL-1492) we e main ained in DMEM supplemen ed wi h 20% e al bo ine se um, 4 mM glu amine and 1% ol/ ol penicillin/s ep omycin a 37oC. P io o ans ec ion, cells we e pla ed in 6-well pla es a a densi y o 106 cells pe well and we e g own in he p esence o 100 nM dexame hasone o 48 h. T ansduc ion wi h adeno i us we e pe o med in 1 mL Op iMEM con aining 100 nM dexame hasone using he indica ed i al concen a ions. 2.4 Collec ion o Cul u e Media and Cells Se en y- wo hou s a e ans ec ion, condi ioned media om HEK 293A cells we e wi hd awn and subjec ed o 200 g × 5 min cen i uga ion o ob ain cell ee condi ioned media. Cells we e washed wice gen ly wi h ice-cold PBS and washed o he pla es in 1.5 ml o PBS and cen i uged a 200 g × 5 min. Fo whole cell lysa es, he esul an cell pelle s we e esuspended in 300 μl o 1 × Laemmli bu e , ollowed by 3 × 15 sec sonica ion. O he wise, cells we e esuspended in 300 μl o RIPA bu e (150 mM NaCl, 1.0% Nonide P-40, 0.5% sodium deoxychola e, 0.1% SDS, and 50 mM T is, pH 8.0) supplemen ed wi h p o ease and phospha ase inhibi o s and incuba ed on ice o 10 min, ollowed by 3 × 5 sec sonica ion. The cell lysa es we e cla i ied by cen i uga ion a 16,000 g × 20 min a 4 °C, and he supe na an s we e designa ed as soluble lysa e ac ion. The pelle s we e washed wice wi h ice-cold PBS and ollowed by 16,000 g × 5 min. The inal pelle s we e esuspended in 100 μl o 1 × Laemmli bu e and sonica ed un il no pa icle was isible. This pa was designa ed as insoluble lysa e ac ion. Condi ioned media om AR42J cells we e ha es ed 24 h a e ansduc ion. Cells we e washed wice wi h PBS, sc aped om he issue cul u e pla es in 1 ml PBS and cen i uged o 10 min a 850 g. Cells we e esuspended in 200 μl Repo e Lysis Bu e (P omega, Madison, 6 WI) and 2 μl Hal P o ease and Phospha ase Inhibi o Single-Use Cock ail (#78442, Pie ce - The mo Scien i ic, Rock o d, IL) was added. Cells we e incuba ed on ice o 15 min and he lysa e was clea ed by cen i uga ion o 10 min a 16,000 g. Fo all cell lysa e samples, he p o ein concen a ion was measu ed wi h he Mic o BCA P o ein Assay Ki (Pie ce - The mo Scien i ic). 2.5 Measu emen o PNLIP p o ein sec e ion om AR42J Cells P o eins in he condi ioned media (40 µl om AR42J cells) we e p ecipi a ed wi h 10% ichlo oace ic acid ( inal concen a ion), esuspended in 20 µl Laemmli sample bu e con aining 100 mM di hio h ei ol, hea -dena u ed a 95oC o 5 min and elec opho esed on 15% SDS- polyac ylamide gels. Gels we e s ained wi h Coomassie Blue. 2.6 Lipase Ac i i y Assay Lipase ac i i y o human PNLIP was de e mined a 25 °C by using he s anda d 5-min pH- s a me hod wi h he emulsion o ibu y in (114 mM) in a o al olume o 15 ml. The eac ions we e ca ied ou in he p esence o absence o o e 5- old mola excess o colipase a 4 mM sodium au odeoxychola e (NaTDC) concen a ion. Fi y µl o condi ioned medium om HEK 293A cells o 100 µl o condi ioned medium om AR42J cells we e added o he eac ion. Fo he assay o he soluble cell lysa e om HEK 293A cells, 100 µl ( o al olume o 300 µl) was added o he eac ion. Lipase ac i i ies a e exp essed as μmoles o a y acid eleased pe min. 2.7 P o ein Immunoblo Condi ioned media and cell lysa es om HEK 293A cells we e ea ed wi h 2 × Laemmli sample bu e supplemen ed wi h 2-me cap oe hanol. A e being hea ed o 95 °C o 5 min, he p epa ed condi ioned medium (20 µl) and cell lysa e samples (10-30 µg o whole cell lysa e and soluble ac ion and 5-10 µg o insoluble ac ion) we e esol ed on 4-15% Mini-P o ean TGX 7 gels (Bio-Rad, He cules, CA) and ans e ed on o an Immobilon-P memb ane (Millipo e, Bed o d, MA). Fo AR42J cells, condi ioned media (0.1 µl) and cell lysa es (0.25 µg o al p o ein o His- ag de ec ion, 2.5 µg o al p o ein o α- ubulin de ec ion o 5 µg o al p o ein o PNLIP de ec ion) we e ea ed as abo e and elec opho esed on 15% T is-glycine minigels and ans e ed on o an Immobilon-P memb ane. A e blocking wi h 5% non- a milk in PBS supplemen ed wi h 0.1% Tween 20, he memb ane was incuba ed wi h p ima y an ibody a 4° C o e nigh and hen wi h seconda y an ibody o 1 h a oom empe a u e. The bands we e de ec ed using he Supe Signal Wes Fem o Maximum Sensi i i y Subs a e o he Supe Signal Wes Pico Chemiluminescen Subs a e (Pie ce - The mo Scien i ic, Rock o d, IL). An ibodies used in his s udy we e as ollowing: abbi polyclonal an ibody agains human PNLIP (1:5,000) was p oduced in ou labo a o y (17); abbi an i-BiP (3177; 1:1,000), GRP94 (2104; 1:1,000), calnexin (2679; 1:2,000), cal e iculin (12238; 1:2,000), and abbi an i-β ubulin (2128; 1:5,000) and GAPDH (5174; 1:5,000) we e pu chased om Cell Signaling (Dan e s, MA); mouse monoclonal an i-α ubulin an ibody (T5168; 1:2,000) was om Sigma-Ald ich (S . Louis, MO), and ho se- adish pe oxidase (HRP)-conjuga ed mouse monoclonal pen a-His an ibody (34460; 1:2,000) was pu chased om Qiagen (Louis ille, KY). The goa an i- abbi IgG conjuga ed wi h ho se adish pe oxidase (31460; 1: 50,000) was pu chased om Pie ce - The mo Scien i ic. 2.8 RT-PCR and Real-Time PCR Analysis To al RNA was isola ed om HEK 293A cells ans ec ed wi h gi en plasmid DNAs using TRIzol (In i ogen), and 1.5 μg o RNA was hen e e se- ansc ibed wi h Moloney mu ine leukemia i us e e se ansc ip ion ki om Ambion (Aus in, TX) a 44° C o 1 h in a 20-μl eac ion olume. Semiquan i a i e measu emen s we e pe o med by PCR o he X-box binding p o ein-1 (XBP1) cDNA and i s spliced o m, using he ollowing p ime s ha ampli y bo h o ms as ollows: XBP1 o wa d p ime , 5′-CCT TGT AGT TGA GAA CCA GG-3′, and XBP1 e e se 8 p ime , 5′-GGG CTT GGT ATA TAT GTG G-3′. The XBP1 p ime s ampli y 441- and 415-bp amplicons om he unspliced and spliced cDNAs, espec i ely. As an endogenous con ol, a 261-bp agmen o GAPDH was also ampli ied, using he ollowing p ime s: GAPDH sense p ime , 5′-GTC CAC TGG CGT CTT CAC CA-3′, and GAPDH an isense p ime , 5′-GTG GCA GTG ATG GCA TGG AC-3′. PCR p oduc s we e un on 2% aga ose gels, and he bands we e isualized by e hidium b omide s aining. To al RNA was ex ac ed om AR42J cell lysa es using he RNeasy Plus Mini ki (Qiagen). RNA was e e se- ansc ibed using High Capaci y cDNA Re e se T ansc ip ion Ki (Applied Biosys ems, G and Island, NY). XBP1 splicing was s udied by PCR using a p ime se o wa d: 5’- GCT TGT GAT TGA GAA CCA GG -3’, e e se: 5’- AGG CTT GGT GTA TAC ATG G -3’) ha lanked he spliced egion and ampli ied bo h spliced (421 bp) and unspliced (447 bp) o ms. The PCR p oduc s we e esol ed on 2% aga ose gels and s ained wi h e hidium b omide. Exp ession o mRNA o BiP and cal e iculin was measu ed by eal ime PCR (7500 Real Time PCR Sys em, Applied Biosys ems) using TaqMan p ime s wi h TaqMan Uni e sal PCR Mas e mix (Applied Biosys ems). Gene exp ession was quan i a ed using he compa a i e CT me hod (ΔΔCT me hod). Th eshold cycle (CT) alues we e de e mined wi h he 7500 Sys em Sequence De ec ion So wa e 1.3. Exp ession le els o a ge genes we e i s no malized o he GAPDH in e nal con ol gene (ΔCT) and hen o exp ession le els measu ed in cells in ec ed wi h 2×107 p u/mL con ol ( ec o only) adeno i us (ΔΔCT). Resul s we e exp essed as old changes calcula ed wi h he o mula 2-ΔΔCT. 9 mouse, an age when PNLIP is no exp essed (21, 24). I is plausible ha ca boxyl es e lipase and PNLIPRP2 compensa e o he loss o PNLIP in he PNLIP-de icien mice. In he case o hese b o he s, i appea s ha PNLIPRP2 and CEL canno compensa e o he loss o PNLIP and he esul is s ea o hea. The eason o his may lay in he ma ked di e ences in enzyme kine ics be ween he mouse and human PNLIPRP2 lipases (21, 25). In pa icula he speci ic ac i i y o mouse PNLIPRP2 agains long-chain iglyce ides, he majo die a y a , is abou 7- old g ea e han he ac i i y o human PNLIPRP2 e en unde op imal condi ions (21, 26). In ac , he speci ic ac i i y o mouse PNLIPRP2 agains long chain iglyce ides is only sligh ly lowe han he speci ic ac i i y o mouse PNLIP. Fu he mo e, mouse PNLIPRP2 has 10- old highe speci ic ac i i y agains sho and medium chain iglyce ides han human PNLIPRP2. The highe speci ic ac i i y may allow mouse PNLIPRP2 o adequa ely compensa e o he absence o PNLIP in PNLIP-de icien mice. 4.4 ER s ess esponse The second impo an inding o ou s udy is he up- egula ion o he ER s ess esponse in cells exp essing p.T221M PNLIP. A obus ER s ess esponse can ac i a e cell-dea h pa hways, which could po en ially damage he panc eas. This mechanism has been sugges ed o o he mis olding a ian s o PRSS1, CTRC and CPA1 (27-29). Each o hese a ian s inc eases ER s ess in ans ec ed cells and a e isk ac o s o ch onic panc ea i is in humans. The accumula ion o mis olded p.T221M PNLIP could also inc ease he isk o ch onic panc ea i is. In he epo o he wo b o he s, he e a e sugges ions ha hey had panc ea ic insu iciency and no jus PNLIP de iciency. In addi ion o s ea o hea, low se um ca ni ine and low se um i amin E le els, bo h b o he s had low ecal elas ase le els and he p oband had an abno mal panc eolau yl es . Nei he b o he had e idence o an in es inal mucosal de ec ha migh impai a y acid abso p ion. No adiog aphic imaging o he panc eas was epo ed so i is no known i he b o he s’ panc eas had ana omic abno mali ies consis en wi h ch onic 16 panc ea i is. E en so, he a ailable da a sugges s ha he p.T221M PNLIP may ha e b oade a ec s no limi ed o a diges ion. Al hough he p.T221M PNLIP mu a ion has only been epo ed in a single consanguineous amily, he possibili y ha o he mu a ions in PNLIP inc ease he isk o panc ea ic disease should be conside ed. 17 Acknowledgemen s: This wo k was suppo ed by Na ional Ins i u es o Heal h G an s R01DK097241 and R01DK080820 o M.E.L. and R01DK082412, R01DK058088, and R01DK095753 o M.S-T. 18 REFERENCES 1. Figa ella, C., A. De Ca o, D. Leupold, and J. R. Poley. 1980. Congeni al panc ea ic lipase de iciency. J Pedia 96: 412-416. 2. Figa ella, C., G. A. Neg i, and H. Sa les. 1972. P esence o colipase in a congeni al panc ea ic lipase de iciency. Biochim Biophys Ac a 280: 205-211. 3. Ligumsky, M., E. G ano , D. B anski, H. S ankiewicz, and R. Golds ein. 1990. Isola ed lipase and colipase de iciency in wo b o he s. Gu 31: 1416-1418. 4. La b e, F., E. Ha emann, J. B. Co on, M. Ma hieu, A. Cha a , and P. Mo eau. 1969. [Ch onic dia hea due o panc ea ic lipase de iciency]. Pedia ie 24: 807-813. 5. Ghishan, F. K., J. R. Mo an, P. R. Du ie, and H. L. G eene. 1984. Isola ed congeni al lipase-colipase de iciency. Gas oen e ology. 86: 1580-1582. 6. Sheldon, W. 1964. Congeni al Panc ea ic Lipase De iciency. A ch Dis Child 39: 268-271. 7. Rey, J., J. F ezal, P. Roye , and M. Lamy. 1966. [Congeni al absence o panc ea ic lipase]. A chi es ancaises de pedia ie 23: 5-14. 8. Ve ge , P., R. Babin, J. M. Guilla d, J. P. Babin, P. Cixous, and J. L. Laigle. 1971. [Ch onic s ea o hea in child en due o a congeni al insu iciency o panc ea ic lipase]. A chi es ancaises de pedia ie 28: 992. 9. Mulle , D. P., J. P. McCollum, R. S. T ompe e , and J. T. Ha ies. 1975. P oceedings: S udies on he mechanism o a abso p ion in congeni al isola ed lipase de iciency. Gu 16: 838. 10. Balze , E. 1967. Angebo ene Mangel an Pank easlipase. Z Gas oen e ol 5: 239-246. 11. Hegele, R. A., D. D. Ramda h, M. R. Ban, M. N. Ca u he s, C. V. Ca ing on, and H. Cao. 2001. Polymo phisms in PNLIP, encoding panc ea ic lipase, and associa ions wi h me abolic ai s. J Hum Gene 46: 320-324. 12. Beha , D. M., L. Basel-Vanagai e, F. Glase , M. Kaplan, S. Tzu , N. Magal, T. Eidli z- Ma kus, Y. Haimi-Cohen, G. Sa ig, C. Bo mans, M. Shoha , and A. Zeha ia. 2014. Iden i ica ion 19 o a no el mu a ion in he PNLIP gene in wo b o he s wi h congeni al panc ea ic lipase de iciency. J Lipid Res 55: 307-312. 13. Ca ie e, F., C. Wi he s-Ma inez, H. an Tilbeu gh, A. Roussel, C. Cambillau, and R. Ve ge . 1998. S uc u al basis o he subs a e selec i i y o panc ea ic lipases and some ela ed p o eins. Biochim Biophys Ac a 1376: 417-432. 14. an Tilbeu gh, H., Y. Ga gou i, C. Dezan, M. P. Eglo , M. P. Nesa, N. Ruganie, L. Sa da, R. Ve ge , and C. Cambillau. 1993. C ys alliza ion o panc ea ic p ocolipase and o i s complex wi h panc ea ic lipase. J Mol Biol 229: 552-554. 15. Winkle , F. K., A. D'A cy, and W. Hunzike . 1990. S uc u e o human panc ea ic lipase. Na u e 343: 771-774. 16. Jennens, M. L., and M. E. Lowe. 1995. The C- e minal domain o human panc ea ic lipase is equi ed o s abili y and maximal ac i i y bu no colipase eac i a ion. J. Lip. Res. 36: 1029-1036. 17. Yang, Y., and M. E. Lowe. 1998. Human panc ea ic iglyce ide lipase exp essed in yeas cells: pu i ica ion and cha ac e iza ion. P o ein Exp Pu i 13: 36-40. 18. Gilham, D., E. D. Labon e, J. C. Rojas, R. J. Jandacek, P. N. Howles, and D. Y. Hui. 2007. Ca boxyl es e lipase de iciency exace ba es die a y lipid abso p ion abno mali ies and esis ance o die -induced obesi y in panc ea ic iglyce ide lipase knockou mice. J Biol Chem 282: 24642-24649. 19. Huggins, K. W., L. M. Cama o a, P. N. Howles, and D. Y. Hui. 2003. Panc ea ic iglyce ide lipase de iciency minimally a ec s die a y a abso p ion bu d ama ically dec eases die a y choles e ol abso p ion in mice. J Biol Chem 278: 42899-42905. 20. D' Agos ino, D., R. A. Co dle, J. Kullman, C. E lanson-Albe sson, L. J. Muglia, and M. E. Lowe. 2002. Dec eased pos na al su i al and al e ed body weigh egula ion in p ocolipase de icien mice. J. Biol. Chem. 277: 7170-7177. 20 21. Xiao, X., L. E. Ross, R. A. Mille , and M. E. Lowe. 2011. Kine ic p ope ies o mouse panc ea ic lipase- ela ed p o ein-2 sugges he mouse may no model human a diges ion. J Lipid Res 52: 982-990. 22. Li, X., S. Lindquis , M. Lowe, L. Noppa, and O. He nell. 2007. Bile sal -s imula ed lipase and panc ea ic lipase- ela ed p o ein 2 a e he domina ing lipases in neona al a diges ion in mice and a s. Pedia Res 62: 537-541. 23. Lowe, M. E., M. H. Kaplan, L. Jackson-G usby, D. D'Agos ino, and M. J. G usby. 1998. Dec eased neona al die a y a abso p ion and T cell cy o oxici y in panc ea ic lipase- ela ed p o ein 2-de icien mice. J Biol Chem 273: 31215-31221. 24. D'Agos ino, D., and M. E. Lowe. 2004. Panc ea ic lipase- ela ed p o ein 2 is he majo colipase-dependen panc ea ic lipase in suckling mice. J Nu 134: 132-134. 25. Ama a, S., D. La on , B. Fio en ino, P. Boullange , F. Ca ie e, and A. De Ca o. 2009. Con inuous measu emen o galac olipid hyd olysis by panc ea ic lipoly ic enzymes using he pH-s a echnique and a medium chain monogalac osyl diglyce ide as subs a e. Biochim Biophys Ac a. 26. Xiao, X., A. Mukhe jee, L. E. Ross, and M. E. Lowe. 2011. Panc ea ic lipase- ela ed p o ein-2 (PLRP2) can con ibu e o die a y a diges ion in human newbo ns. J Biol Chem 286: 26353-26363. 27. Ke esz u i, E., R. Szmola, Z. Kuko , P. Simon, F. U. Weiss, M. M. Le ch, and M. Sahin- Tó h. 2009. He edi a y panc ea i is caused by mu a ion-induced mis olding o human ca ionic ypsinogen: a no el disease mechanism. Hum Mu a 30: 575-582. 28. Szmola, R., and M. Sahin-Tó h. 2010. Panc ea i is-associa ed chymo ypsinogen C (CTRC) mu an elici s endoplasmic e iculum s ess in panc ea ic acina cells. Gu 59: 365-372. 29. Wi , H., S. Bee , J. Rosendahl, J. M. Chen, G. R. Chandak, A. Masamune, M. Bence, R. Szmola, G. O acz, M. Macek, J ., E. Bha ia, S. S eigenbe ge , D. Lashe , F. Bühle , C. Delapo e, J. Tebbing, M. Ludwig, C. Pilsak, K. Saum, P. Buge , E. Masson, S. Paliwal, S. 21 Bhaska , A. Sobczynska-Tomaszewska, D. Bak, I. Balascak, G. Choudhu i, D. N. Reddy, G. V. Rao, V. Thomas, K. Kume, E. Nakano, Y. Kaku a, T. Shimosegawa, L. Du ko, A. Szabó, A. Schnú , P. Hegyi, Z. Rakonczay, J ., R. P ü ze , A. Schneide , D. A. G onebe g, M. B aun, H. Schmid , U. Wi , H. F iess, H. Algül, O. Land , M. Schuelke, R. K üge , B. Wiedenmann, F. Schmid , K. P. Zimme , P. Ko acs, M. S um oll, M. Blühe , T. Mülle , A. Janecke, N. Teich, R. G ü zmann, H. U. Schulz, J. Mössne , V. Keim, M. Löh , C. Fé ec, and M. Sahin-Tó h. 2013. Va ian s in CPA1 a e s ongly associa ed wi h ea ly onse ch onic panc ea i is. Na Gene . FIGURE LEGENDS Fig. 1. Sec e ion o PNLIP and p.T221M PNLIP om ans ec ed HEK 293A cells. The media om ans ec ed cells was sampled 72 h a e ans ec ion. (A) The lipase ac i i y agains ibu y in in 50 µl o medium was measu ed in he s anda d 5 minu e pH-s a assay. The mean and SD o 3 sepa a e de e mina ions is plo ed. (B) The p esence o PNLIP was de e mined by SDS PAGE and p o ein immunoblo wi h he abbi an i-human PNLIP an ise a ollowed by de ec ion wi h he HRP conjuga ed goa an i- abbi IgG an ise a. (C) The ela i e amoun s o lipase in he samples we e de e mined by densi ome y o 3 sepa a e expe imen s. Fig. 2. The in acellula accumula ion o PNLIP and p.T221M PNLIP in ans ec ed HEK 293A cells. Se en y- wo hou s a e ans ec ion he cells we e ha es ed and p ocessed o o al, soluble and insoluble ac ions as desc ibed in Ma e ials and Me hods. The p esence o PNLIP was de e mined by SDS PAGE and p o ein immunoblo wi h he abbi an i-human PNLIP an ise a ollowed by de ec ion wi h he HRP conjuga ed goa an i- abbi IgG an ise a. The ela i e amoun s o lipase in he samples we e de e mined by densi ome y. The mean and SD o 3 sepa a e de e mina ions is plo ed. (A) The o al amoun o PNLIP and p.T221M PNLIP a e 22 shown. GAPDH se es as he loading con ol. (B) The amoun o each lipase in he soluble in acellula ac ion is shown. (C) The amoun o each lipase in he insoluble in acellula ac ion is shown. ***P ≤ 0.001. Fig. 3. Exp ession o PNLIP and p.T221M PNLIP in adeno i us ansduced AR42J cells. The cells we e ansduced wi h h ee di e en i e s o adeno i us. (A) Aliquo s o condi ioned medium we e assayed by SDS PAGE ollowed by Coomassie Blue s aining (uppe panel) o by p o ein immunoblo wi h he HRP-conjuga ed mouse monoclonal pen a-His an ibody (middle panel) o we e assayed o lipase ac i i y agains ibu y in in 100 µl o medium in he s anda d 5 minu e pH-s a assay (bo om panel). The mean and SD o 3 sepa a e de e mina ions is plo ed. (B) Aliquo s o o al cell lysa e we e analyzed by SDS PAGE and p o ein immunoblo wi h he HRP-conjuga ed mouse monoclonal pen a-His an ibody. α- ubulin was used as a loading con ol. (C) AR42J cells we e ans ec ed wi h 108 p u adeno i us and he o al, soluble and insoluble ac ions we e analyzed by p o ein immunoblo . ***P ≤ 0.001 Fig. 4. E ec o exp ession o p.T221M PNLIP in HEK 293A cells on ER s ess. Cellula lysa es o ans ec ed HEK 293A cells we e p epa ed as desc ibed in Ma e ials and Me hods. A leas h ee measu emen s om sepa a e expe imen s we e done in each case. (A) The ela i e amoun s o BiP de e mined by p o ein immunoblo and quan i a ed by densi ome y. (B) The ela i e amoun s o cal e iculin de e mined by p o ein immunoblo and quan i a ed by densi ome y. (C) The ela i e amoun s o GRP94 de e mined by p o ein immunoblo and quan i a ed by densi ome y. (D) To al RNA was isola ed om ans ec ed HEK 293A cells. The amoun o XBP1 mRNA splicing was measu ed by RT-PCR, sepa a ion by aga ose gel elec opho esis, e hidium b omide s aining and densi ome y. The uppe panel is a ep esen a i e aga ose gel o he RT-PCR p oduc s. The bo om panel is he amoun o XBP1 mRNA splicing quan i a ed by densi ome y. **P ≤ 0.01. 23 Fig. 5. E ec o exp ession o p.T221M PNLIP in AR42J cells on ER s ess. The cells we e ansduced wi h h ee di e en i e s o each adeno i us cons uc . To al RNA was isola ed om he cells as desc ibed. The amoun o mRNA encoding BiP and cal e iculin was de e mined by eal- ime RT-PCR and he amoun o XBP1 mRNA splicing de e mined by RT-PCR, sepa a ion by aga ose gel elec opho esis, e hidium b omide s aining and densi ome y. (A) Mean and SD o he ela i e BiP le els. (B) Mean and SD o he ela i e cal e iculin le els. (C) Amoun o XBP1 mRNA splicing is gi en. The uppe panel shows a ep esen a i e aga ose gel sepa a ion o he RT-PCR p oduc s. The bo om panel p esen s he mean and SD o 3 expe imen s. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 24 Mock WT T221M Rela i e P o ein Le els 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Mock WT T221M Fa y Acid Released (moles pe Minu e) 0.0 0.5 1.0 1.5 2.0 2.5 A. B. C. Mock WT T221M 150 50 100 75 37 26 kDa Figu e1