Tài liệu Báo cáo khoa học: Hu-K4 is a ubiquitously expressed type 2 transmembrane protein associated with the endoplasmic reticulum ppt

Seibel, Zara Hashemol Hosseini and Wolfgang Hampe Center of Experimental Medicine, Institute of Biochemistry and Molecular Biology II: University Hospital Eppendorf, Hamburg, Germany The

protein associated with the endoplasmic reticulum

Antonia Munck, Christopher Bo¨hm, Nicole M Seibel, Zara Hashemol Hosseini and

Wolfgang Hampe

Center of Experimental Medicine, Institute of Biochemistry and Molecular Biology II: University Hospital Eppendorf, Hamburg, Germany

The Hu-K4 protein was first identified as a human

homologue of the K4L protein of vaccinia virus [1]

K4L is a nonessential protein in the life cycle of the virus

and has unknown function Both Hu-K4 and K4L

con-tain two HXKXXXXD⁄ E (HKD) motifs which make

them members of the superfamily of HKD proteins

together with phospholipase D proteins and

phospholi-pid synthases [2] The closest homologues of Hu-K4 are

found in other mammals, murine SAM9 [3] has 93%

identical amino acid residues (Fig 1) More distantly

related proteins are found in Xenopus (54%) and

Dro-sophila(48%) and in vaccinia virus In addition to the

viral K4L protein (48%) this virus also encodes the

clo-sest relative of Hu-K4 with known function, the most

abundant viral protein p37 (21%) Other members of

the HKD superfamily are the phospholipase D

iso-forms Like the other proteins shown in Fig 1 they

harbour two HKD motifs which are involved in the

catalytic process [4] For this reason Hu-K4 was named phospholipase D3 in the GenBank entry NP_036400 although outside the HKD motifs no similarity exists Phospholipase D enzymes catalyse the hydrolysis of membrane phospholipids, e.g of phosphatidyl choline

to choline and phosphatidic acid which was ascribed

a second-messenger function Two isoforms, phospho-lipase D1 and D2, are well characterized and part of different signalling cascades implicated in membrane trafficking, cytoskeletal reorganization, receptor endo-cytosis, exoendo-cytosis, cell migration, and regulation of the cell cycle [5] For the murine orthologue of Hu-K4, SAM9, so far no phospholipase D activity could be assigned indicating that Hu-K4 and SAM9 might have another function [3]

The above mentioned protein p37 is essential for efficient cell-to-cell spreading by vaccinia virus [6] During maturation of the virus, p37 is required for the

Keywords

topology, subcellular localization, gene

structure, expression pattern, translational

control

Correspondence

W Hampe, Institut fu¨r Biochemie und

Molekularbiologie II, Molekulare Zellbiologie,

Universita¨tsklinikum Eppendorf, Martinistr.

52, D-20246 Hamburg, Germany

Fax: +49 40 42803 4592

Tel: +49 40 42803 9967

E-mail: hampe@uke.uni-hamburg.de

(Received 2 December 2004, revised 1

February 2005, accepted 8 February 2005)

doi:10.1111/j.1742-4658.2005.04601.x

Hu-K4 is a human protein homologous to the K4L protein of vaccinia virus Due to the presence of two HKD motifs, Hu-K4 was assigned to the family of Phospholipase D proteins although so far no catalytic activity has been shown The Hu-K4 mRNA is found in many human organs with highest expression levels in the central nervous system We extended the ORF of Hu-K4 to the 5¢ direction As a consequence the protein is 53 amino acids larger than originally predicted, now harbouring a putative transmembrane domain The exon⁄ intron structure of the Hu-K4 gene reveals extensive alternative splicing in the 5¢ untranslated region Due to the absence of G⁄ C-rich regions and upstream ATG codons, the mRNA isoform in brain may be translated with higher efficacy leading to a high Hu-K4 protein concentration in this tissue Using a specific antiserum pro-duced against Hu-K4 we found that Hu-K4 is a membrane-bound protein colocalizing with protein disulfide isomerase, a marker of the endoplasmic reticulum Glycosylation of Hu-K4 as shown by treatment with peptide N-glycosidase F or tunicamycin indicates that Hu-K4 has a type 2 trans-membrane topology

Abbreviations

EST, expressed sequence tag; GST, glutathione S-transferase; Hu-K4, human K4L homologue; PNGaseF, peptide N-glycosidase F.

wrapping of infectious intracellular mature virions by cisternae derived from virus-modified trans-Golgi or endosomal membranes to form intracellular enveloped virions [7] The integrity of the p37 HKD motifs is required for the formation of the intracellular en-veloped virion membrane [8] During this process p37 shuttles between plasma membrane and intracellular organelles [9] and is involved in the trafficking of integral membrane proteins from the Golgi apparatus This function is inhibited by a phospholipase D inhib-itor although overexpressed phospholipase D cannot complement a p37 deficient virus Therefore, an important role in inducing the formation of vesicle precursors of the vaccinia virus membrane via phospho-lipase D activity or activation was predicted [10] Nevertheless, although p37 exhibits phospholipase C and A activities toward a variety of lipid substrates,

no phospholipase D activity could be detected in vitro [11] Despite the homology between p37 and Hu-K4 or K4L no function could so far be assigned to the K4 proteins

In this paper we characterize Hu-K4 We describe the correction of the originally proposed ORF, the identification of splice variants, and the determination

of the expression pattern using mRNA hybridization and a new specific antiserum By performing a glycosy-lation analysis we prove Hu-K4 to be a type 2 trans-membrane protein

Results and Discussion

mRNA-expression pattern of Hu-K4

In a Northern blot we identified at least two different transcript sizes for the Hu-K4 mRNA (Fig 2A) A short variant of about 1700 nucleotides is abundantly present in brain Lower amounts of this variant and also of a longer isoform of about 2200 nucleotides were ubiquitiously expressed with lowest expression levels in leukocytes To check the mRNA expression in other tissues, we hybridized a multiple-tissue expres-sion array (Fig 2B) which confirms the notion that the Hu-K4 mRNA is most highly expressed in brain, but

at a lower level in almost all tissues These data are in agreement with those of Pedersen et al [3], who found the mRNA of the murine homologue SAM9 mainly in brain, but also in other tissues In situ mRNA hybrid-ization showed a neuronal expression in the adult and developing murine brain [3] The human multiple-tissue expression array shows a weak signal for Hu-K4

in the corpus callosum, which contains mainly glial but no neuronal cell bodies, indicating that also in the human brain mainly neurons express Hu-K4

Hum MKP KLMYQELKVPAEEPANELPMNEIEAWKAAEKKARWVLLVLILAVVGFGAL.MTQL

Mur MKP KLMYQELKVPVEEPAGELPLNEIEAWKAAEKKARWVLLVLILAVVGFGAL.MTQL

Xen MSS KVEYKPIQ.PHEEAENHFLQHELHKVKA.RKYYRCALVVAIIITLVFCIL.ASQL

K4L MNPDNTIA

dro MPEYKKLEDQESDVENANRTTVQNTATVQDAGEGQRQAAGQQAGQMVTVSLFMLLFLGSS

p37 M

Hum FLWEYGDLHLFGP N QRPAPCYDPCEAVLVESIPEGLDFPNASTGNPSTSQAWLG

Mur FLWEYGDLHLFGP N QRPAPCYDPCEAVLVESIPEGLEFPNATTSNPSTSQAWLG

Xen LLFPFLSITSQTT ETVLNKDIRCDDQCRFVLVESIPEGLVYDANSTINPSIFQSWMN

K4L VITETIPIGMQFDKV YLSTFNMWRE

Dro YFQPRPRLHQYKGGRGHGLLEK FD.CNIQLVESIPIGLTYPDGSPRFLSTYEAWLE

p37 WPFASVPA GAKC RLVETLPENMDFRSD HLTTFECFNE

Hum LLAGAHSSLDIASFYWTLTNNDTHT.QEPSAQQGEEVLRQLQTLAPKG VNVRIAV

Mur LLAGAHSSLDIASFYWTLTNNDTHT.QEPSAQQGEEVLQQLQALAPRG VKVRIAV

Xen IITNAKSSIDIASFYWSLTNEDTQT.KEPSAHQGELILQELLNLKQRG VSLRVAV

K4L ILSNTTKTLDISSFYWSLSD EVGTNFGTIILNEIVQLPKRG VRVRVAV

Dro LLESATTSLDIASFYWTLKAEDTPGVSDNSTRPGEDVFARLLANGNGGSRSPRIKIRIAQ

p37 IITLAKKYIYIASFC CNPLSTTRGALIFDKLKEASEKG IKIIVLL

Hum SKPSGPQPQADLQALLQS.GA.QVRMVDMQK.LTHGVLHTKFWVVDQTHFYLGSANMDWR

Mur SKPNGP LADLQSLLQS.GA.QVRMVDMQK.LTHGVLHTKFWVVDQTHFYLGSANMDWR

Xen NPPDSPIRSKDISALKDR.GA.DVRVVDMPK.LTDGILHTKFWVVDNEHFYIGSANMDWR

K4L NKSNKPLKDVER LQM AGVEVRYIDITNILG.GVLHTKFWISDNTHIYLGSANMDWR

Dro SEPSSGTPNLNTKLLASA.GAAEVVSISFPKYFGSGVLHTKLWVVDNKHFYLGSANMDWR

p37 DERGKR NLGELQSHCPDINFITVNIDKKNNVGLLLGCFWVSDDERCYVGNASFTGG

Hum SLTQVKELGVVMYNCSCLARDLTKIFEAYWFLGQAGSSIPSTWPRFYDTRYNQETPMEIC

Mur SLTQVKELGVVMYNCSCLARDLTKIFEAYWFLGQAGSSIPSTWPRSFDTRYNQETPMEIC

Xen SLTQVKELGATIYNCSCLAQDLKKIFEAYWILGLPNATLPSPWPANYSTPYNKDTPMQVM

K4L SLTQVKELGIAIFNNRNLAADLTQIFEVYWYLG VNNLPYNWKNFYPSYYNTDHPLSIN

Dro ALTQVKEMGVLVQNCPELTHDVAKIFGEYWYLGNSESSRIPDWDWRYATSYNLKHPMQLS

p37 SIHTIKTLGV.YSDYPPLATDLRRRFDTF KAFNSAKNSWLNLCSAACCLPVSTAYH

Hum LNGTPAL.AYLASAPPPLCPSGRTPDLKALLNVVDNARSFIYVAVMNYLPTLEFSHPHR.

Mur LNGTPAL.AYLASAPPPLCPSGRTPDLKALLNVVDSARSFIYIAVMNYLPTMEFSHPRR.

Xen LNSTASQ.VYLSSSPPPLSATGRTDDLQSIMNIIDDAKKFVYISVMDYSPTEEFSHPRR.

K4L VSGVP.HSVFIASAPQQLCTMERTNDLTALLSCIRNASKFVYVSVMNFIPII.YSKAGKI

Dro VNKNTSIEGFLSSSPPPLSPSGRTDDLNAILNTINTAITYVNIAVMDYYPLIIYEKNHH.

p37 IKN.PIGGVFFTDSPEHLLGYSRDLDTDVVIDKLRSAKTSIDIEHLAIVPTTRVD GNS

Hum FWPAIDDGLRRATYERGVKVRLLISCWGHSEPSMRAFLLSLAALRDNHTHSDIQVKLFV

Mur FWPAIDDGLRRAAYERGVKVRLLISCWGHSDPSMRSFLLSLAALHDNHTHSDIQVKLFV

Xen YWPEIDNHLRKAVYERNVNVRLLISCWKNSRPSMFTFLRSLAALHSNTSHYNIEVKIFV

K4L LFWPYIEDELRRSAIDRQVSVKLLISCWQRSSFIMRNFLRSIAMLKSKN IDIEVKLFI

Dro YWPFIDDALRKAAVERGVAVKLLISWWKHSNPSEDRYLRSLQDLASKEDKIDIQIRRFI

p37 YYWPDIYNSIIEAAINRGVKIRLLVGNWDKNDVYSMATARSLDALC VQNDLSVKVFT

Hum VPADEAQARIPYARVNHNKYMVTERA.TYIGTSNWSGNYFTETAGTSLLVTQNGRGG

Mur VPTDESQARIPYARVNHNKYMVTERA.SYIGTSNWSGSYFTETAGTSLLVTQNGHGG

Xen VPATEAQKKIPYARVNHNKYMVTDRV.AYIGTSNWSGDYFINTAGSALVVNQTQSAGTSD

K4L VP DADPPIPYSRVNHAKYMVTDKT.AYIGTSNWTGNYFTDTCGASINITPDDGLG

Dro VPTDSSQEKIPFGRVNHNKYMVTDRV.AYIGTSNWSGDYFTDTAGIGLVLSETFETETTN

p37 IQ NNTKLLIVDDEYVHITSANFDGTHYQNHGFVSF NSIDK

Hum LRSQLEAIFLRDWDSPYSHDLDTSADSVGNACRLL

Mur LRSQLEAVFLRDWESPYSHDLDTSANSVGNACRLL

Xen TIQMQLQTVFERDWNSNYSLTFNTLSSWKEK.C.IF

K4L LRQQLEDIFMRDWNSKYSYEL YDTSPTKRCKLLKNMKQCTNDIYCDEIQPEKEIPEY

Dro TLRSDLRNVFERDWNSKYATPL V

p37 QLVSEAKKIFERDWVSSHSKSLKI

541

K4L SLE

Hum B

A

Mur Xen K4L Dro p37

Fig 1 Homology of Hu-K4 with other members of the HKD

super-family (A) Protein alignment performed with the CLUSTAL method [23].

Highly conserved residues found in at least five of the six proteins are

boxed The two HKD motifs are overlined (B) Phylogenetic tree of

the alignment in (A) Hum, human Hu-K4 (AAH36327); Mur, murine

SAM9 (AAC73069); Xen, Xenopus laevis MGC68676 (AAH59981);

K4L, Vaccinia virus K4L (NP_063673); Dro, Drosophila melanogaster

CG9248-PA (NP_724313); p37, Vaccinia virus p37 (P20638).

Gene structure of Hu-K4 The Hu-K4 gene is located on human chromosome 19q13.2 and entirely covered by the BAC clone CTC-492K19 with the GenBank accession number AC010271 We sequenced the image-cDNA clone

159455 (GenBank H15746 and H15747) and identified

an ORF encoding a protein of 490 amino acids (Fig 3A) This is in agreement with the GenBank entry BC036327⁄ AAH36327, whereas the original GenBank entry for human Hu-K4 (U60644) predicted

an N-terminally truncated protein due to a missing nucleotide in codon 52 leading to a shift in the ORF The alignment of several dozen sequences of expressed sequence tags (EST) clones gave no indication for fur-ther cDNAs with a missing nucleotide Several puta-tive in-frame ATG start codons are present close to the 5¢ end of the Hu-K4 cDNA behind an in-frame stop codon at nucleotide 321 (Table 1) None of them corresponds to the optimal context for the initiation of translation given by Kozak [12], since none of the four possible start codons has a G in position +4, only the first ATG has a purine in position )3 We therefore assume that translation usually starts at position 330 Nevertheless, we cannot exclude a leaky scanning by the small ribosomal subunit [12] leading to N-termin-ally truncated Hu-K4 isoforms The additional 53 N-terminal amino acids, which are not present in the original database entry, are highly homologous to Hu-K4 from mouse (GenBank BC076586) and rat (XM_341811) indicating a high evolutionary pressure

on this sequence and supporting the hypothesis that this part of the mRNA is translated

Eleven exons encode the ORF of human Hu-K4 (Fig 4A, exon 5–15) The analysis of more than 100 GenBank EST clones did not reveal any alternative splicing in the ORF or in the 3¢ untranslated region (UTR) To explain the splice variants observed in the Northern blot we analysed the 5¢-UTR of the available several dozen EST clones which turned out to be highly variable (Fig 4B) Two out of these cDNA clones, both derived from the same adult female breast cDNA library, start with exon 2, all other clones start with exon 1 but skip exon 2 indicating that there might

be two different promotors The clones containing exon 1 are very diverse in their exon composition before exon 5 They might or might not bear the exons

3 or 4, part or the entire region between exons 3 and 4 (Fig 4B, 3¢ and 4¢), or extended exons 1 or 5 (Fig 4B, 1¢ and 5¢) and therefore differ in size Most often clones with 44, 258 or 422 bp extensions between exons 1 and 5 are found, nicely explaining the mRNA isoforms seen in the Northern blot (Fig 2A) The

brainheartskelet

al muscle colon thymus spleen kidney liversmall

intestine placent

a lungleukocytes

2200 bp A

B

1700 bp

1 2

A

H

G

D

F

E

A

B

C

A

H

G

D

F

E

A

B

C

Fig 2 Hu-K4 mRNA distribution (A) Northern blot analysis using a

32

P-labelled Hu-K4 cDNA fragment (B) Human multiple tissue

expression array.

short isoform, which is abundantly expressed in brain,

probably includes exons 1⁄ 1¢ ⁄ 5–10 as 10 out of the 15

respective clones (67%) originate from the fetal or

adult central nervous system In contrast, only 15%

and 20% of the clones with the 258 or 422 bp

exten-sions are derived from brain, respectively

The different 5¢-UTRs might function in

transla-tional control In most cases, 5¢-UTRs that enable

effi-cient translation are short, have a low GC content and

do not contain upstream ATG codons [13] The

lon-gest isoform of the Hu-K4 mRNA is 457 bp longer

than the shortest version Exon 4 is the largest exon, alone accounting for 214 nucleotides Especially exons 1¢, 4¢ and 4 have a high G ⁄ C content (Table 2) Upstream ATG codons are found in exons 1, 2, 3, 4¢ and 4, but only those in exons 3, 4¢ and 4 are located

in an adequate context for translational start (Table 2) Taken together, these data suggest that the smaller mRNA variant from brain which lacks exons

2, 3, 3¢, 4, 4¢ and 5¢ might be more efficiently trans-lated than the larger isoforms which predominate in other tissues

0 -3

B A

*

Fig 3 Hu-K4 mRNA and protein (A) Hu-K4

cDNA and amino acid sequence derived

from the image clone 159455 (GenBank

H15746) Putative start codons of the ORF

and the preceding in-frame stop codon are

boxed The two HKD motifs are underlined

in bold, the putative transmembrane domain

is marked with a dotted line The C-terminal

prenylation motif is marked in grey, the

polyadenylation signals are labelled with

lines on top and the N-glycosylation motifs

are enclosed in ovals The two peptides

used for antibody production are underlined

with a thin line The exons of the 5¢ UTR

are given according to Fig 4 (B)

Hydrophi-licity plot The arrow indicates the putative

transmembrane domain.

In contrast to the 5¢-UTR, the 3¢-UTR of the

Hu-K4 mRNA seems to be the same in all EST clones

The poly(A) tail starts about 300 nucleotides behind

the stop codon Two putative polyadenylation signals

(AATAAG and AATAAC) are present about 20

nucleotides in front of the poly(A) tail (Fig 3A) Both

are not identical to the most often used signal AAT

AAA which is found in 65% of human mRNAs, but

especially variants with a single pyrimidine substitution

like AATAAC also seem to be functional [14]

Expression and topology of the Hu-K4 protein

In order to raise an antiserum against the Hu-K4

pro-tein two rabbits were injected with a mixture of the

two peptides underlined in Fig 3 One of the rabbits

produced an antiserum staining a 65-kDa band in

a western blot of Hu-K4-transfected COS-7 cells,

whereas the respective preimmune serum was negative

(Fig 5A) Preincubation of the antiserum with the

C-terminal peptide LDTSADSVGNACRLL alone or

with both peptides used for immunization prevented

staining, indicating that the antiserum recognizes the

carboxy tail of Hu-K4 The apparent molecular mass

of 65 kDa is higher than the calculated molecular mass

of 55 kDa for Hu-K4 hinting at a post-translational

modification, e.g glycosylation, of Hu-K4 in cultured

cells The absence of additional bands in the western

blot indicates that at least in COS-7 cells only the first

start codon (Table 1) is used

As the Hu-K4 mRNA is most abundant in brain

and since the small mRNA isoform found in brain is

probably translated with highest efficiency, we chose

membranes from rat brains to check whether the

Hu-K4 antiserum recognizes endogenous Hu-K4

Indeed, a 55 kDa protein was identified by western

blotting which was stained by the antiserum only in

the absence of the peptides used for immunization

Other proteins were also stained by the saturated anti-serum (Fig 5B) The sequence of the peptides used for immunization is highly conserved from human to mouse and rat and the antiserum recognized Hu-K4 in human, rat and mouse brain (not shown)

To identify the subcellular distribution of Hu-K4 we analysed transiently transfected COS-7 cells by immuno-cytochemistry Extensive colocalization with protein disulfide isomerase hints at a localization in the endoplasmic reticulum (Fig 6) although an obvious retrieval signal is missing

The human phospholipases D1 and D2 are mainly associated with the plasma membrane or with the membranes of intracellular organelles although they lack a transmembrane domain They are attached to the cytoplasmic face of the membranes via palmitoyl anchors [15] as is the vaccinia virus protein p37 [16] Hu-K4 also partitioned exclusively to the membrane fraction after a crude membrane preparation, whereas the soluble protein fraction and conditioned medium were devoid of immunoreactivity (Fig 5C) There are two possible means by which Hu-K4 could be attached

to membranes: First, similar to PLD1 and PLD2, Hu-K4 could be a cytosolic protein anchored to the cytoplasmic face of the membrane by C-terminal prenylation as predicted by psort ii The C-terminal leucine residue in the prenylation motif (Fig 3A) hints

at a geranylgeranyl anchor Alternatively, Hu-K4 could harbour a transmembrane domain formed by a stretch

of 17 hydrophobic amino acids (Figs 3A and B; psort

ii predicts a transmembrane domain but not a cleaved signal peptide) Since several basic amino acids are present N terminal to the hydrophobic stretch but none on the C-terminal side, the first 38 amino acid residues are expected to be cytoplasmic, whereas the large C-terminal domain including the two HXKXXXXD⁄ E-motifs would be luminal or extracel-lular [17] This domain inherits seven putative glycosy-lation sites which could only be glycosylated if it enters the endoplasmic reticulum The N-terminal 38 amino acid residues lack consensus sites for N-glycosylation (Fig 3A) To differentiate between the two topologies,

we deglycosylated Hu-K4 heterologously produced in COS-7 cells (Fig 5D) Indeed, we found a reduction in the apparent molecular mass of Hu-K4 after treatment with peptide N-glycosidase F (PNGaseF) showing that Hu-K4 is a type 2 transmembrane protein These data are confirmed by a reduced molecular mass of Hu-K4

in cells that have been grown in the presence of the glycosylation inhibitor tunicamycin (Fig 5D) Differ-ential glycosylation also explains the different apparent molecular masses found for Hu-K4 in cultured cells and brain

Table 1 Putative start codons of Hu-K4 Comparison of the optimal

translational start site given by Kozak [12] and the putative start

co-dons in the Hu-K4 mRNA The most important nucleotides of the

Kozak consensus sequence are indicated in bold A weak context

means that none of the important nucleotides indicated in bold is

present, an adequate sequence comprises only one, a strong

con-text both [13].

Position

Kozak consensus A

The two HXKXXXXD⁄ E motifs of Hu-K4 are

positioned in the luminal domain whereas those of

PLD1 and PLD2 are located to the cytosol If

Hu-K4 can hydrolyse phospholipids, it will therefore

use lipids of the opposite membrane leaflet as

substrates

Experimental procedures

Hybridization

Commercially available human multiple tissue Northern blot and multiple tissue expression array (Clontech) were

A

B

Fig 4 Exon–intron structure of the Hu-K4 gene (A) Position of the exons on the BAC clone CTC-492K19 (GenBank AC010271.8) The first and the last nucleotide of each exon are given The positions of the polyadenylation site, the stop codon and the putative start codons ATG1 and ATG 4 are indicated The size of exons 1–5 is drawn in scale (B) Alternative splicing of the 5¢-UTR The splice variants, the number of nucleotides between exon 1 and exon 5 (size) and the number of expressed sequence tags relating to each variant (#EST) are given.

hybridized using a [32P]-labelled human Hu-K4 probe

com-prising the first 916 nucleotides shown in Fig 3A as

des-cribed [18]

Bioinformatics

DNA and protein analysis were performed using the

pro-gram dnastar Hu-K4 encoding ESTs were identified in

GenBank using the basic local alignment tool (blast) of

the National Centre for Biotechnology Information (http://

www.ncbi.nlm.nih.gov/BLAST) Analysis of transmembrane

domains and protein motifs was carried out using psort ii (http://psort.nibb.ac.jp)

Antibody production, western blotting and immunocytochemistry

Two rabbits were immunized with a mixture of the follow-ing two human Hu-K4 peptides: NH2-LDTSADSVG NACRLL-COOH (coupled to keyhole limpet haemocyanin using glutaraldehyde) and NH2 -CTWPRFYDTRYNQETP-CONH2 (coupled to keyhole limpet haemocyanin at the

Table 2 Exons encoding the 5¢ UTR of Hu-K4 For each exon its length, G ⁄ C content, and, if present, ATG codons with position (numbering

as in AC010271.8), context and size of the encoded peptide are given.

Kozak consensus A

44013

CCA ATG A CGC ATG C

Weak Weak

> 54 aa

9 aa

60933 60939

GTA ATG C TGC ATG T TCC ATG G G

Adequate Weak Adequate

13 aa

33 aa

31 aa

105

kDa

15

50

105

kDa 15

50

35 75

αHu-K4

PIS

Peptide

+ - +

- +

- +

αHu-K4

Peptide

+ +

- +

105

kDa 15

50 35 75

Hu-K4 mock Med Sol Mem Sol Mem

105

kDa

50 35 75 Con PNGase - + Tunicamyc. - +

Fig 5 Hu-K4 protein expression (A) Characterization of the Hu-K4 antiserum Lysates of COS-7 cells transiently transfected with the Hu-K4 cDNA were analysed by western blotting using the Hu-K4 antiserum (1 : 2000) or the respective preimmune serum (PIS, 1 : 2000) Preincu-bation of the antiserum with the C-terminal peptide used for immunization inhibited labelling of Hu-K4 (B) Detection of endogenous Hu-K4 Membranes from rat brain were analysed by western blotting using the Hu-K4 antiserum in the absence or presence of the C-terminal pep-tide used for immunization (C) Hu-K4 is membrane bound COS-7 cells transfected with the Hu-K4 cDNA or with vector alone (mock) were disrupted by sonification, separated into a membrane (Mem) and a soluble (Sol) fraction by ultracentrifugation and analysed by western blot-ting using the Hu-K4 antiserum In the first lane a blot of conditioned medium of Hu-K4-transfected cells is shown (D) Deglycosylation Membranes from COS-7 cells transfected with the Hu-K4 cDNA were incubated in the absence (–) or presence (+) of PNGaseF As a control (Con) nontreated membranes are shown The two lanes on the right show the western blot analysis of Hu-K4 from membranes of trans-fected COS-7 cells growing for 24 h in the absence (–) or presence (+) of the N-glycosylation inhibitor tunicamycin (1 lgÆmL)1).

cysteine residue) After several injections one rabbit

pro-duced an antiserum appropriate for western blotting and

immunocytochemistry

Western blotting and immunocytochemistry were

per-formed as described [19] using rabbit anti-(Hu-K4) Ig

(1 : 2000 for western blot, 1 : 1000 for ICC) or mouse

anti-(protein disulfide isomerase) Ig (1 : 100, StressGen) To

prove specificity, the diluted Hu-K4 antiserum was

incuba-ted with the indicaincuba-ted peptides ( 10 lgÆmL)1) for 1 h at

37
C prior to incubation

Heterologous expression, sample preparation

and deglycosylation

The image-cDNA clone 159455 (GenBank H15746 and

H15747) encoding full-length Hu-K4 was supplied by the

RZPD Deutsches Ressourcenzentrum fu¨r Genomforschung

[20] For heterologous expression the Hu-K4 ORF was

cloned into pcDNA3.1⁄ Hygro (Invitrogen)

COS-7 cells were cultured and transfected by

electro-poration as described [21] To prevent N-glycosylation

tu-nicamycin was added at a concentration of 1 lgÆmL)1 to

the growth medium

Conditioned medium was prepared 48 h after

electropo-ration by incubating cells for 16 h in a minimal amount of

medium For western blot analysis, transfected cells were

lysed using 50 mm Tris, 150 mm NaCl, 2 mm EDTA, 1%

(v⁄ v) NP-40, pH 7.6, unsolubilized material was removed

by centrifugation Cell membranes were prepared by

ultra-sonification and differential centrifugation at 1000 g and

100 000 g

Brain membranes were prepared using an Ultra-Turrax blender, a Teflon homogenizer and differential centrifuga-tion as described [19]

For PNGaseF digestion, membranes from Hu-K4-trans-fected COS-7 cells ( 30 lg protein) were suspended in sample buffer [2% (w⁄ v) SDS, 5% (v ⁄ v) 2-mercaptoetha-nol, 12% (v⁄ v) glycerol, 50 mm Tris pH 6.8] and heated to

95
C for 5 min Then, the samples were diluted 20-fold with buffer A [0.5% (v⁄ v) Triton X-100, 10 mm EDTA,

20 mm NaH2PO4 pH 7.4] and heated again to 95
C for

5 min After addition of 30 U PNGaseF (Roche) or an equivalent volume buffer A, deglycosylation was allowed to proceed for 10–14 h gently agitated at 37
C After a sec-ond addition of PNGaseF or buffer A, the incubation was repeated Proteins were then precipitated using methanol⁄ chloroform [22], separated by SDS⁄ PAGE and Hu-K4 detected by western blotting

Acknowledgements

We thank Prof Ulrike Beisiegel and Prof Chica Schal-ler for discussion and providing the laboratory equip-ment and Susanne Hoppe for technical assistance This work was supported by the Deutsche Forschungsge-meinschaft (SFB 444 B10)

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Fig 6 Hu-K4 immunocytochemistry COS-7 cells were transiently

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(C,D), or with anti-(protein disulfide isomerase) and anti-mouse Cy3

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