Filtrin is a novel member of nephrin-like proteins

Pekka Ihalmo, Tuula Palm�een, Heikki Ahola, Elsa Valtonen, and Harry Holth€oofer*

Department of Bacteriology and Immunology, and Biomedicum Molecular Medicine, Haartman Institute, University of Helsinki,

and Helsinki University Central Hospital, University of Helsinki, PB 63 (Haartmaninkatu 8), Helsinki FIN-00014, Finland

Received 18 November 2002

Abstract

NPHS1 encodes nephrin, the core protein of the interpodocyte slit diaphragm of the kidney glomerulus. NPHS1 is the causative

gene for congenital nephrotic syndrome of the Finnish type (CNF) with massive, treatment resistant proteinuria. We report here the

establishment of a novel nephrin-like gene, NLG1 encoding filtrin, a protein with substantial homology to human nephrin. Filtrin is

a type I transmembrane protein consisting of 708 amino acids. Together with the recently cloned NEPH1, NLG1 establishes a new

nephrin-like subgroup of genes belonging to the immunoglobulin superfamily of cell adhesion molecules. The RNA dot blot ex-

periment revealed that the NLG1 mRNA expression is widely distributed but most prominently observed in the pancreas and lymph

nodes. The expression of NLG1 mRNA in kidney glomeruli was verified with RT-PCR. Further immunoblotting studies with

antifiltrin antibody showed a specific band at 107 kDa in the human and rat glomeruli. In immunofluorescence microscopy specific

staining of glomeruli but also proximal and distal parts of the nephron was seen in human kidney cortex. Due to its structural

similarity and sequence homology as well as partially consistent expression pattern with nephrin we propose that filtrin belongs to a

functionally important complex of proteins of the glomerular filtration barrier.

� 2002 Elsevier Science (USA). All rights reserved.

Keywords: In silico cloning; Glomerulus; Nephrin; NEPH1; Slit diaphragm; Kidney

The kidney glomerular filtration barrier is crucial for

maintaining the water and electrolyte balance of the

body without losing circulating proteins into the urine.

The barrier consists of the porous vascular endothelial

cells, a particular layered basement membrane (GBM)

and the epithelial cells, podocytes [1]. The recent reve-

lations of molecules specific for the podocytes and par-

ticularly for the inter-podocyte slit diaphragms (SD)have suggested that the podocyte SD complex is the key

element in kidney diseases manifesting with proteinuria

[2].

The milestone finding of NPHS1, the gene causing

the lethal congenital nephrotic syndrome of the Finnish

type (CNF) presenting with massive, treatment resistant

proteinuria, has firmly suggested that the respective

protein product, nephrin, forms the dynamic structuralcore component of the SD [3,4]. Nephrin is a trans-

membrane protein of the immunoglobulin superfamily

of adhesion molecules. It is associated with lipid rafts,

mediates outside–in signalling [5,6], and is proposed to

interact in a heterophilic manner with NEPH1 [7,8]. The

intracellular part of nephrin associates with CD2AP

[9,10], a protein initially demonstrated for T cell func-

tions, and with another podocyte specific protein,

podocin [11]. Via these binding partners nephrin is

suggested to interact with the actin cytoskeleton [12]. Adirect functional role for nephrin in disease pathogen-

esis has been shown in the experimental animal models

of, e.g., minimal change disease [13] and diabetic ne-

phropathy [14] as well as in human glomerular diseases

[15].

Here, we report the in silico cloning and the sub-

sequent experimental characterization of a new distinct

gene and protein with substantial homology to nephrin.This new nephrin-like gene 1 (NLG1) encodes a trans-

membrane protein termed filtrin and is prominently

expressed in the lymph nodes, pancreas as well as in

kidney glomerulus, the exclusive sites also expressing

nephrin.

Biochemical and Biophysical Research Communications 300 (2003) 364–370

www.elsevier.com/locate/ybbrc

BBRC

* Corresponding author. Fax: +358-9-191 25501.

E-mail address: harry.holthofer@helsinki.fi (H. Holth€oofer).

0006-291X/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0006-291X(02)02854-1

Materials and methods

In silico cloning. Database searching was performed at the National

Center for Biotechnology Information (NCBI) using the Basic Local

Alignment Search Tool Algorithm (BLAST) [16] (http://

www.ncbi.nlm.nih.gov/BLAST). The human nephrin protein (Gen-

Bank Accession No. AAC39687) was used as a query sequence in the

screening of the non-redundant (nr) and the Expressed Sequence Tag

(EST) databases.

The best candidates were further characterized with bioinformatic

tools accessible via the Internet. Briefly, potential protein domains and

sites were predicted using InterPro [17] (http://www.ebi.ac.uk/interpro/

scan.html) and Prosite [18] (http://tw.expasy.org/tools/scanprosite/)

databases and servers. The O-glycosylation status was analyzed with

NetOGlyc [19] (http://www.cbs.dtu.dk/services/NetOGlyc/). Trans-

membrane localization data were searched using TMHMM [20] (http://

www.cbs.dtu.dk/services/TMHMM-2.0/) and a potential signal peptide

was searched with SignalP [21] (http://www.cbs.dtu.dk/services/Sig-

nalP-2.0/). Multiple sequence alignments were made with ClustalW

[22] (http://www.ebi.ac.uk/clustalw/) and colored with BoxShade

(http://www.ch.embnet.org/software/BOX_form.html).

Primary chromosomal localization data were obtained from the

UniGene database (http://www.ncbi.nlm.nih.gov/UniGene/). The gene

structure was further characterized with pairwise BLAST searches

against the genomic clone RP11-38C1 (AC022315) and Genscan [23]

program (http://genes.mit.edu/GENSCAN.html). The promoter area

was predicted with Proscan [24] (http://bimas.dcrt.nih.gov/molbio/

proscan/) and the CpG-pattern with CpGPlot [25] (http://www.ebi.a-

c.uk/emboss/cpgplot). AUG_EVALUATOR[26] (http://www.itba.-

mi.cnr.it/webgene/) program was used to evaluate the translation

starting codons and the 50 untranslated region in the cDNA.

The cDNA clone DKFZp564A1164 from a human fetal brain li-

brary was obtained from RZPD Resource Center (Berlin, Germany)

for further experiments. Briefly, the library was enriched for full-length

cDNAs using Capfinder protocol [27] (Clontech Laboratories, Palo

Alto, CA, USA). The 2976-bp fragment with a poly(A) tail was cloned

directionally into pAMPI (Life Technologies BRL, Paisley, UK) using

NotI and SalI sites of the vector.

Human tissues and cell culture. Human kidney tissues were from

cadaver donors as previously described [4]. All procedures were ap-

proved by the Ethics Committee of the Helsinki University Central

Hospital.

Human podocyte cell line was provided by Dr. Hermann Pa-

venst€aadt (Department of Medicine, Division of Nephrology, Univer-

sity Hospital of Freiburg, Freiburg, Germany). The generation of this

cell line has been described earlier in detail [28]. Briefly, isolated nor-

mal human glomeruli were suspended in DMEM containing 10% heat-

inactivated FCS, 2.5mM glutamine, 0.1mM sodium pyruvate, 5mM

HEPES buffer, 1mg/ml streptomycin, 100U/ml penicillin, 0.1� non-

essential amino acids (100�; all Seromed, Berlin, Germany), insulin,transferrin, and a 5mM sodium selenite supplement and incubated at

37 �C and 5% CO2 in air. Cell colonies sprouted around the glomeruli

were excised and incubated in 5ml of 0.2% collagenase IV (Sigma–

Aldrich, Deisenhofen, Germany) at 37 �C for 30min followed by wa-

shes and further plating. Cells showed epithelial morphology and

stained positive for Wilm�s tumor antigen (WT1) and nephrin that are

podocyte specific markers in the adult kidney. Cells were negative for

an endothelial cell marker, factor 8 related antigen.

Reverse transcriptase-polymerase chain reaction (RT-PCR) and

sequencing. Total RNA was extracted from isolated glomerular frac-

tions and used as a starting material in the cDNA synthesis as de-

scribed earlier [29,30]. Briefly, total RNA was extracted from tissues

with Trizol reagent (Life Technologies) according to manufacturer�sinstructions. RNA was treated with RNase-free DNaseI (Promega,

Madison, WI) for 30min at 37 �C together with human placental

RNase inhibitor (Promega) for the removal of residual genomic DNA.

Complementary DNA was prepared from RNA with the M-MLV

reverse transcriptase (Promega) using oligo dT15 primers (Roche Di-

agnostic GmbH, Mannheim, Germany). Control reactions were car-

ried out without the reverse transcriptase enzyme.

A NLG1 cDNA specific primer pair was designed, a sense primer

50-TTA GGC CCG TGG AGC TAG A-30 (nucleotides 464–482) and

an antisense primer 50-CAT CTC GGA ACC ACA GCA AT-30 (nu-

cleotides 685–666), both from the sequence encoding the extracellular

part of filtrin. As a control, b-actin was amplified with a sense primer

50-AAC CGC GAG AAG ATG ACC CAG ATC ATG TTT-30 (nu-

cleotides 353–352) and an antisense primer 50-AGC AGC CGT GGC

CAT CTC TTG CTC GAA GTC-30 (nucleotides 674–703). The am-

plification reactions were carried out using AmpliTaq Gold DNA

polymerase (Applied Biosystems, Foster City, CA) with initial dena-

turation of cDNA at 95 �C 10min, followed by 35 amplification cycles

(94 �C 45 s, 56 �C 1min and 72 �C 45 s), and a final elongation at 72 �Cfor 7min.

The PCR products were gel-purified (QIAquick PCR Purification

kit, Qiagen, Hilden, Germany) and sequenced with an ABI 373 se-

quencer using ABI Prism Dye Terminator kit (Applied Biosystems).

The clone DKFZp564A1164 was sequenced using the same method.

Human tissue RNA dot blot. Human Multiple Tissue Expression

(MTE) Array (Clontech Laboratories) containing poly(A) mRNAs

from 76 tissues was used to determine the NLG1 expression profile.

The RNA dot blot was hybridized with [32P]dCTP-labeled probes

synthesized from NLG1 cDNA (nucleotides 496–2273). Briefly, the

insert was digested out from the above-mentioned plasmid with NdeI

(New England Biolabs, Beverly, MA) restriction enzyme, and gel-pu-

rified, after which 25 ng of it was used as a template. Complementary

DNA probes were made with Rediprime II random prime labeling

system (Amersham Pharmacia Biotech, Uppsala, Sweden) and subse-

quently purified with a NICK column (Amersham Pharmacia Biotech)

according to manufacturer�s instructions. The blot was hybridized

according to manufacturer�s instructions and analyzed after an over-

night exposure with a Bio-imaging analyzer (Fuji Photo Film,

Kanakawa, Japan).

Production of antipeptide antibodies. A sequence-specific extracel-

lular oligopeptide from the clone DKFZp564A1164 representing the

amino acids 21–40 with an additional C-terminal cysteine was selected,

synthesized, and purified by Alpha Diagnostics International (San

Antonio, TX). For immunizations the peptide was coupled to an an-

tigenic carrier protein Keyhole Limpet Hemocyanin (KLH).

The antibodies were produced according to standard protocols.

Briefly, rabbits were immunized with 500lg antigen in Freund�scomplete adjuvant (Difco Laboratories, Detroit, MI). Two booster

injections with 400lg antigen in Freund�s incomplete adjuvant were

given at intervals of 4 weeks and the serum was collected. Peptide-

specific fractions were further immunoaffinity purified on Epoxy-Ac-

tivated Sepharose CL-6B Resin (Sigma–Aldrich) coupled to the cor-

responding peptide.

Immunoblotting. Human and rat glomeruli and cultured human

podocytes were homogenized in reducing Laemmli buffer, boiled for

5min, and run under reducing conditions using 7.5% Tris–HCl Ready

Gels (Bio-Rad Laboratories, Hercules, CA) in Protean Mini-gel elec-

trophoresis system (Bio-Rad Laboratories). The proteins were elec-

trotransferred to nitrocellulose membranes (Amersham Life Sciences,

Buckinghamshire, England) and unspecific binding was blocked by

incubating overnight at 4 �C in 3% skimmed milk in PBS. Filters were

incubated for an hour with diluted anti-filtrin antibody (15 lg/ml) andwashed with PBS–0.2% Triton X-100 followed by an hour of incuba-

tion with the horseradish peroxidase-conjugated goat anti-rabbit IgG

(Jackson Immunoresearch Laboratories, West Grove, PA; 1:20,000).

All antibody dilutions were made in 1% skimmed milk in PBS. After

washes with PBS–0.2% Triton X-100, the bound antibodies were de-

tected with SuperSignal West Pico Chemiluminescent Substrate kit

(Pierce, Rockford, IL).

P. Ihalmo et al. / Biochemical and Biophysical Research Communications 300 (2003) 364–370 365

Immunofluorescence. Normal human cortical tissue frozen sections

were fixed with 3.5% paraformaldehyde for 20min and thoroughly

washed with PBS. The sections were further treated with 0.1% Triton

X-100 in PBS for 15min, washed, incubated for 1 h with anti-filtrin

antibody (0.16mg/ml), and washed. Next, the sections were further

incubated for 1 h with FITC-conjugated goat anti-rabbit IgG (H+L)

(Jackson Immunoresearch Laboratories) diluted at 1:100. All antibody

dilutions were done in PBS including 5% normal human serum and

0.1% Triton X-100. After the final wash the sections were embedded in

Immu-Mount medium (Shandon, Pittsburgh, PA).

Results and discussion

The zipper model of the interdigitating podocyte foot

processes by Rodewald and Karnovsky [31] suggests the

maintainence of a tight seal impermeable to macro-

molecules with a free passage of water, electrolytes, and

small molecules into the urine. With the milestone

finding of NPHS1 [3], encoding nephrin, a completely

new understanding of the molecular complex of the

functional glomerular filtration barrier has rapidlyemerged. While maintaining dynamic outside–in sig-

naling and adhesive functions by the extracellular do-

main [6], the main protein partners of nephrin including

podocin [32], CD2AP [9], and densin [48] most likely

compose a functional protein complex at the level ofthe SD.

We used the BLAST algorithm at the NCBI to search

for uncharacterized clones and EST sequences homol-

ogous to human nephrin and the best candidates, in-

cluding the clone DKFZp564A1164, were analyzed

further. The characterization of the NLG1 cDNA

(XM_048304) revealed that the gene product of NLG1,

termed filtrin (XP_048304), is a type I transmembraneprotein with an extracellular region containing five

tandem immunoglobulin-like domains, a 23-amino acid

a-helical transmembrane region, and a 178-residue cy-

toplasmic domain with a proline-rich region (Fig. 1).

Similar to the domain structure of NEPH1 [8], the Ig-

like domains Ig1, Ig2, Ig3, and Ig5 of filtrin belong to

the C2-subtype of immunoglobulin-like domains and

the Ig3 is similar to the polycystic kidney disease protein

Table 1

Alignment of the N-terminal parts of human nephrin-like proteins

FILTRIN 1 .........................MLRMRVPALLVLLFCFRGRAGPSPH.FLQQPEDLVVLLGEEARLPCALGAYWGLVQWTKSGLALGGQRDLNEPH1 1 ............................MLSLLVWILTLSDTFSQGTQTR.FSQEPADQTVVAGQRAVLPCVLLNYSGIVQWTKDGLALGMGQGLKIAA1867 1 GMKPFQLDLLFVCFFLFSQELGLQKRGCCLVLGYMAKDKFRRMNEGQVYS.FSQQPQDQVVVSGQPVTLLCAIPEYDGFVLWIKDGLALGVGRDLNEHPRIN 1 ..................MALGTTLRASLLLLGLLTEGLAQLAIPASVPRGFWALPENLTVVEGASVELRCGVSTPGSAVQWAKDGLLLGPDPRI

FILTRIN 70 PGWSRYWISGNAANGQHDLHIRPVELEDEASYECQATQAG....LRSRPAQLHVLVPPEAPQVL..GGPSVSLVAGVPANLTCRSRGDARPTPELNEPH1 67 KAWPRYRVVGSADAGQYNLEITDAELSDDASYECQATEAA....LRSRRAKLTVLIPPEDTRID..GGPVILLQAGTPHNLTCRA.FNAKPAATIKIAA1867 95 SSYPQYLVVGNHLSGEHHLKILRAELQDDAVYECQAIQAA....IRSRPARLTVLVPPDDPVIL..GGPVISLRAGDPLNLTCHA.DNAKPAASINEHPRIN 78 PGFPRYRLEGDPARGEFHLHIEACDLSDDAEYECQVGRSEMGPELVSPRVILSILVPPKLLLLTPEAGTMVTWVAGQEYVVNCVS.GDAKPAPDI

FILTRIN 159 LWFRDGVLLDGATFHQTLLKEGTPGSVESTLTLTPFSHDDGATFVCRARSQALPTGRDTAITLSLQYPPEVTLSASPHT....VQEGEKVIFLCQNEPH1 155 IWFRDGTQQEGAVASTELLKDGKRETTVSQLLINPTDLDIGRVFTCRSMNEAIPSGKETSIELDVHHPPTVTLSIEPQT....VQEGERVVFTCQKIAA1867 183 IWLRKGEVINGATYSKTLLRDGKRESIVSTLFISPGDVENGQSIVCRATNKAIPGGKETSVTIDIQHPPLVNLSVEPQP....VLEDNVVTFHCSNEHPRIN 172 TILLSGQTISDISANVNEGSQQKLFTVEATARVTPRSSDNRQLLVCEASSPALEAPIKASFTVNVLFPPGPPVIEWPGLDEGHVRAGQSLELPCV

FILTRIN 250 ATAQPPVTGYRWAKGGSPVLGARGPRLEVVADASFLTEPV.........SCEVSNAV..GSANRSTALDVLFGPILQAKPEPVSVDVGEDASFSCNEPH1 246 ATANPEILGYRWAKGGFLIEDAHESRYETNVDYSFFTEPV.........SCEVHNKV..GSTNVSTLVNVHFAPRIVVDPKPTTTDIGSDVTLTCKIAA1867 274 AKANPAVTQYRWAKRGQIIKEASGEVYRTTVDYTYFSEPV.........SCEVTNAL..GSTNLSRTVDVYFGPRMTTEPQSLLVDLGSDAIFSCNEHPRIN 267 ARGGNPLATLQWLKNGQPVSTAWGTEHTQAVARSVLVMTVRPEDHGAQLSCEAHNSVSAGTQEHGITLQVTFPPSAIIILGSASQTENKNVTLSC

FILTRIN 334 AWRGNPLPRVTWTRRGGAQVLGSGAT..............LRLPSVGPEDAGDYVCRAEAGLSGLRGGAAEARLTVNAPP....VVTALHSAPAFNEPH1 330 VWVGNPPLTLTWTKKDSNMVLSNSNQ..............LLLKSVTQADAGTYTCRAIVPRIGV..AEREVPLYVNGPP....IISSEAVQYAVKIAA1867 358 AWTGNPSLTIVWMKRGSGVVLSNEKT..............LTLKSVRQEDAGKYVCRAVVPRVGA..GEREVTLTVNGPP....IISSTQTQHALNEHPRIN 362 VSKSSRPRVLLRWWLGWRQLLPMEETVMDGLHGGHISMSNLTFLARREDNGLTLTCEAFSEAFTKETFKKSLILNVKYPAQKLWIEGPPEGQKLR

FILTRIN 411 LRGPARLQCLVFASPAPDAVVWSWDEGFLEAGSQGRFLVETFPAPESRGGLGPGLISVLHISGTQESDFSRSFNCSARNRLGEGGAQASLGRR..NEPH1 405 RGDGGKVECFIGSTPPPDRIAWAWKENFLEVGTLERYTVERTNSGS.......GVLSTLTINNVMEADFQTHYNCTAWNSFGPGTAIIQLEER..KIAA1867 433 HGEKGQIKCFIRSTPPPDRIAWSWKENVLESGTSGRYTVETISTEE.......GVISTLTISNIVRADFQTIYNCTAWNSFGSDTEIIRLKEQGSNEHPRIN 457 AGTRVRLVCLAIGGNPEPSLMWYKDSRTV...TESRLPQESRRVHLGSVEKSGSTFSRELVLVTGPSDNQAKFTCKAGQLSASTQLAVQFPPTN.

FILTRIN 504 ..........DLLPTVRIV.AGVAAATTTLLMVITG.VALCCWRHSKASASFSEQKNLMRIPG.SSDGSSSRGPEEEETGSRED...RGPIVHTDNEPH1 491 ..........EVLPVGIIAGATIGASILLIFFFIAL.VFFLYRRRKGSRKDVTLRKLDIKVETVNREPLTMHSDREDDTASVST...ATRVMKAIKIAA1867 521 EMKSGAGLEAESVPMAVIIGVAVGAGVAFLVLMATI.VAFCCARSQRNLKGVVSAKNDIRVEIVHKEPASGREGEEHSTIKQLM...MDRGEFQQNEHPRIN 548 .........VTILANASALRPGDALNLTCVSVSSNPPVNLSWDKEGERLEGVAAPPRRAPFKGSAAARSVLLQVSSRDHGQRVTCRAHSAELRET

FILTRIN 583 HS......DLVLEEKGTLETKDPTNGYYKVRGVSVNEPH1 572 YSSFKDDVDLKQDLRCDTIERPRIRGRLNTSYSD.KIAA1867 612 DSVLKQLEVLKEEEKEFQNLKDPTNGYYSVNTFKENEHPRIN 634 VSSFYRLNVLYRPEFLGEQVLVVTAVEQGEALLPV

Fig. 1. The schematic diagram shows the domain structure of filtrin.

366 P. Ihalmo et al. / Biochemical and Biophysical Research Communications 300 (2003) 364–370

domain, first identified as an Ig-like structural elementof polycystin-1 [33]. Filtrin consists of 708 amino acids

with an estimated molecular mass of 75.1 kDa and with

a pI of 6.48.

The extracellular region of filtrin is preceded by a

cleavable N-terminal signal sequence (amino acids 1–

20). A cell attachment sequence RGD [34], also known

as integrin-recognition site, follows at amino acid posi-

tions 149–151. Three potential N-glycosylation sites atamino acid positions 143, 301, and 484, and four po-

tential O-glycosylation sites at amino acid positions 222,

230, 232, and 364 suggest further post-translational

modification. The cytoplasmic domain has a potential

protein kinase C phosphorylation site at amino acid

position 560, and four casein kinase II phosphorylation

sites at amino acid positions 542, 571, 614, and 638. Due

to potential intracellular phosphorylation proposingsignaling properties filtrin may well complement the SD

structure and participate in its regulation.

The extracellular region of filtrin shows strong se-

quence homology to human nephrin (30%), NEPH1

(44%), and KIAA1867 (41%), coded by another gene

belonging to the subgroup of nephrin-like genes (Table

1). In addition to their sequence homology, these four

proteins are structurally related due to the conserved Ig-like domains, the transmembrane region, and the C-

terminal intracellular region. Interestingly, the intracel-

lular domain of filtrin and the proline-rich region show

very low homology to any other characterized protein.

Proline-rich regions are usually known as ligands for

SH3- and WW domain-containing proteins [35]. In the

kidney glomerulus the most characterized SH3-con-

taining protein is CD2AP that is shown to interact withnephrin [10]. Another ligand in glomerular podocytes

for proline-rich proteins is the tight-junction associated

MAGI-1 that is composed of WW-domains. It is pro-

posed to interact with the podocyte-specific protein

synaptopodin [36]. Therefore, it is tempting to speculate

that filtrin could participate in similar heterophilic in-

teractions defining the SD structure.

The first ATG codon (AUG in the mRNA) at the 50

end of the cDNA was followed shortly by an in-frame

translation stop codon. When proceeding towards the 30

end, another translation starting codon follows starting

a long open reading frame. However, the AUG_E-

VALUATOR program revealed that the first start co-

don is located in a weak AUG context. Therefore, we

presume that the first open reading frame (upstream

ORF) is untranslated and the second one is the mainopen reading frame (main ORF) encoding filtrin. It is

known that the efficiency of the translation reinitiation

from the second starting site is dependent on the size of

the upstream ORF [37]. In the case of the NLG1 mRNA

the upstream ORF is relatively short. However, this

mechanism provides a new regulation layer to control

protein expression levels and timing [38,39].

RNA expression pattern of NLG1

According to RNA dot blot analysis of a panel of

poly(A) mRNA from 72 human tissues the NLG1

mRNA is primarily expressed in the lymph nodes and

the pancreas (Fig. 2). With RT-PCR, NLG1 mRNA was

detected in isolated kidney glomeruli and in a cultured

human podocyte cell line (Fig. 3). The expression ofNLG1 mRNA in the kidney, pancreas, and lymph node

as well as in the eye, lung, brain, and germ cells was also

supported by the EST sequence analysis. Furthermore,

the sequence of DKFZp564A1164 (on GenBank Ac-

cession No. AL136654) carried a sequencing error in a

critical nucleotide position, leading to an artefactual

translation stop codon and a failure to identify the full-

length protein product filtrin.We and others have previously shown that nephrin is

restrictedly expressed in the glomerular podocytes [4], in

the pancreas [40], lymphoid tissue [49], and brain [41].

Interestingly, the expression pattern of NLG1 mRNA

and filtrin is highly similar to that of nephrin. In addi-

tion to the proposed function in kidney filtration, filtrin

may still show additional functions in other tissues.

Mapping of NLG1 in the genome

The NLG1 gene was localized in silico to human

chromosome locus 19q13.1 adjacent toNPHS1 encoding

the transmembrane protein nephrin. It is of particular

Fig. 2. Northern dot blot analysis of human fetal and adult poly(A)

mRNA of 72 different tissues showing distribution and relative ex-

pression of NLG1. A strong signal can be seen at positions B9 and F7

corresponding to poly(A) mRNA from adult pancreas and lymph

nodes.

Fig. 3. Expression of NLG1 mRNA can be demonstrated in kidney

glomerulus (1, RT+; 2, RT)) and cultured podocytes (3, RT+; 4, RT);and 5, H2O) using RT-PCR.

P. Ihalmo et al. / Biochemical and Biophysical Research Communications 300 (2003) 364–370 367

note that NPHS1 and NLG1 are transcribed in opposite

directions and the distance between the transcription

starting points is approximately 5-kb (Fig. 4). The core

NLG1 promoter is TATA- and CAAT-less and it residesin a CpG-island.

Moeller et al. [42] have previously demonstrated that

regulatory elements needed for the endogenous Nphs1

expression in the mouse are located 5.4-kb or even 8.3-

kb upstream of the transcription initiation site. More-

over, the region 1.25-kb upstream is shown to direct

podocyte-specific expression both in human [43] and

mouse [44]. Thus, the promoter regions, although readin opposite directions, of NPHS1 and NLG1 overlap at

least in the pancreas, arising a possibility of finely tuned

regulation of both reading directions.

Splicing variants of NLG1 mRNA

According to sequence alignments, the gene consists

of fifteen exons. In addition to the full-length form, two

alternatively spliced mRNA variants were discovered.The a-form (according to the EST clone AL529115)

lacks the exon No. 4, the first Ig-like domain of the

translated protein thus being truncated. Filtrin-b(BC007312) represents a soluble form consisting only of

the sequence coding for the N- and C-terminal parts

(Fig. 5). In addition to a-nephrin [4], examples of such

soluble transmembrane-free splicing variants include,

e.g., interleukin-6-receptor [45] and T cell receptor [46].The respective cDNA clones encoding the beta-form

were found exclusively in eye-derived EST libraries.

Immunoblotting and immunofluorescence

Immunoblotting studies of the human and rat glo-

merular lysates with the anti-filtrin antibody showed the

identification of a protein band with the apparent mo-

lecular weight of 107 kDa, suggesting post-translationalmodification. In addition, a triple band corresponding

to the molecular weights of 98, 103, and 117 kDa was

observed in the cultured podocyte cell line (Fig. 6). The

immunofluorescence staining with the anti-filtrin anti-

body revealed expression in the kidney glomeruli as well

as to some extent in proximal and distal tubuli (Fig. 7).

Adhesion molecules, like integrins, cadherins, and

immunoglobulin superfamily proteins, mediating cell–

cell or cell–matrix interactions, are of pivotal impor-tance for various physiological functions including glo-

merular filtration [47]. Recently cloned NEPH1 and its

lethally proteinuric knock-out phenotype [8] propose

that nephrin-like molecules of the immunoglobulin su-

perfamily are basic elements in the kidney glomerular

filtration barrier.

We named this new gene NLG1 by its similarity to

nephrin. With the intriguing chromosomal localizationadjacent to but in an opposite direction to NPHS1 and a

shared gene regulation area together with the consistent

tissue distribution pattern with nephrin suggest that

Fig. 5. Structure of the identified NLG1 mRNA splicing variants: full-

length form (A), a-form lacking the exon No. 4 (B), and the soluble b-form (C).

Fig. 6. Demonstration of filtrin protein in human (1) and rat (2)

glomeruli and in cultured human podocytes (3) using immunoblotting.

Fig. 7. Immunostaining of normal human kidney cortex with anti-filtin

antibody shows distinct reactivity of the glomerulus in a podocyte-like

manner. Some reactivity is seen in tubular epithelial cells (magnifica-

tion 280�).

Fig. 4. Genomic organization and exon–intron structure of human

NPHS1 and NLG1 in chromosome locus 19q13.1. The distance be-

tween the transcription starting sites is approximately 5-kb.

368 P. Ihalmo et al. / Biochemical and Biophysical Research Communications 300 (2003) 364–370

filtrin may be a closely associated molecule in themodulation of nephrin properties.

Acknowledgments

We thank Dr. Hermann Pavenst€aadt for the generous supply of

cultured podocyte cells. Eeva H€aayri and Liisa Pirinen are acknowl-

edged for expert technical assistance. Ansa Karlberg is thanked for

secretarial advice. This study was supported by the Sigrid Juselius

Foundation, the Finnish Diabetes Association, the Finnish Kidney

Foundation, the Academy of Finland, Helsinki University Central

Hospital, and the European Union (Grant QLG1-2000-00619).

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