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

Hu-K4 is a ubiquitously expressed type 2 transmembrane 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, exocytosis, 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. 1718 FEBS Journal 272 (2005) 1718–1726 ª 2005 FEBS 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. 1 60 Hum MKP KLMYQELKVPAEEPANELPMNEIEAWKAAEKKARWVLLVLILAVVGFGAL.MTQL Mur MKP KLMYQELKVPVEEPAGELPLNEIEAWKAAEKKARWVLLVLILAVVGFGAL.MTQL Xen MSS KVEYKPIQ.PHEEAENHFLQHELHKVKA.RKYYRCALVVAIIITLVFCIL.ASQL K4L MNPDNTIA dro MPEYKKLEDQESDVENANRTTVQNTATVQDAGEGQRQAAGQQAGQMVTVSLFMLLFLGSS p37 M 61 120 Hum FLWEYGDLHLFGP N QRPAPCYDPCEAVLVESIPEGLDFPNASTGNPSTSQAWLG Mur FLWEYGDLHLFGP N QRPAPCYDPCEAVLVESIPEGLEFPNATTSNPSTSQAWLG Xen LLFPFLSITSQTT ETVLNKDIRCDDQCRFVLVESIPEGLVYDANSTINPSIFQSWMN K4L VITETIPIGMQFDKV YLSTFNMWRE Dro YFQPRPRLHQYKGGRGHGLLEK FD.CNIQLVESIPIGLTYPDGSPRFLSTYEAWLE p37 WPFASVPA GAKC RLVETLPENMDFRSD HLTTFECFNE 121 180 Hum LLAGAHSSLDIASFYWTLTNNDTHT.QEPSAQQGEEVLRQLQTLAPKG VNVRIAV Mur LLAGAHSSLDIASFYWTLTNNDTHT.QEPSAQQGEEVLQQLQALAPRG VKVRIAV Xen IITNAKSSIDIASFYWSLTNEDTQT.KEPSAHQGELILQELLNLKQRG VSLRVAV K4L ILSNTTKTLDISSFYWSLSD EVGTNFGTIILNEIVQLPKRG VRVRVAV Dro LLESATTSLDIASFYWTLKAEDTPGVSDNSTRPGEDVFARLLANGNGGSRSPRIKIRIAQ p37 IITLAKKYIYIASFC CNPLSTTRGALIFDKLKEASEKG IKIIVLL 181 240 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 241 300 Hum SLTQVKELGVVMYNCSCLARDLTKIFEAYWFLGQAGSSIPSTWPRFYDTRYNQETPMEIC Mur SLTQVKELGVVMYNCSCLARDLTKIFEAYWFLGQAGSSIPSTWPRSFDTRYNQETPMEIC Xen SLTQVKELGATIYNCSCLAQDLKKIFEAYWILGLPNATLPSPWPANYSTPYNKDTPMQVM K4L SLTQVKELGIAIFNNRNLAADLTQIFEVYWYLG VNNLPYNWKNFYPSYYNTDHPLSIN Dro ALTQVKEMGVLVQNCPELTHDVAKIFGEYWYLGNSESSRIPDWDWRYATSYNLKHPMQLS p37 SIHTIKTLGV.YSDYPPLATDLRRRFDTF KAFNSAKNSWLNLCSAACCLPVSTAYH 301 360 Hum LNGTPAL.AYLASAPPPLCPSGRTPDLKALLNVVDNARSFIYVAVMNYLPTLEFSHPHR. Mur LNGTPAL.AYLASAPPPLCPSGRTPDLKALLNVVDSARSFIYIAVMNYLPTMEFSHPRR. Xen LNSTASQ.VYLSSSPPPLSATGRTDDLQSIMNIIDDAKKFVYISVMDYSPTEEFSHPRR. K4L VSGVP.HSVFIASAPQQLCTMERTNDLTALLSCIRNASKFVYVSVMNFIPII.YSKAGKI Dro VNKNTSIEGFLSSSPPPLSPSGRTDDLNAILNTINTAITYVNIAVMDYYPLIIYEKNHH. p37 IKN.PIGGVFFTDSPEHLLGYSRDLDTDVVIDKLRSAKTSIDIEHLAIVPTTRVD GNS 361 420 Hum .FWPAIDDGLRRATYERGVKVRLLISCWGHSEPSMRAFLLSLAALRDNHTHSDIQVKLFV Mur .FWPAIDDGLRRAAYERGVKVRLLISCWGHSDPSMRSFLLSLAALHDNHTHSDIQVKLFV Xen .YWPEIDNHLRKAVYERNVNVRLLISCWKNSRPSMFTFLRSLAALHSNTSHYNIEVKIFV K4L LFWPYIEDELRRSAIDRQVSVKLLISCWQRSSFIMRNFLRSIAMLKSKN IDIEVKLFI Dro .YWPFIDDALRKAAVERGVAVKLLISWWKHSNPSEDRYLRSLQDLASKEDKIDIQIRRFI p37 YYWPDIYNSIIEAAINRGVKIRLLVGNWDKNDVYSMATARSLDALC VQNDLSVKVFT 421 480 Hum VPADEAQARIPYARVNHNKYMVTERA.TYIGTSNWSGNYFTETAGTSLLVTQNGRGG Mur VPTDESQARIPYARVNHNKYMVTERA.SYIGTSNWSGSYFTETAGTSLLVTQNGHGG Xen VPATEAQKKIPYARVNHNKYMVTDRV.AYIGTSNWSGDYFINTAGSALVVNQTQSAGTSD K4L VP DADPPIPYSRVNHAKYMVTDKT.AYIGTSNWTGNYFTDTCGASINITPDDGLG Dro VPTDSSQEKIPFGRVNHNKYMVTDRV.AYIGTSNWSGDYFTDTAGIGLVLSETFETETTN p37 IQ NNTKLLIVDDEYVHITSANFDGTHYQNHGFVSF NSIDK 481 540 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). A. Munck et al. Hu-K4 FEBS Journal 272 (2005) 1718–1726 ª 2005 FEBS 1719 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 brain heart skeletal muscle colon thymus spleen kidney liver small intestine placenta lung leukocytes 2200 bp A B 1700 bp 345678910111212 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. Hu-K4 A. Munck et al. 1720 FEBS Journal 272 (2005) 1718–1726 ª 2005 FEBS 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 3 100 B A 200 400300 * 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. A. Munck et al. Hu-K4 FEBS Journal 272 (2005) 1718–1726 ª 2005 FEBS 1721 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 GCC ATG G G Context ATG 1 330 AAG ATG A Adequate ATG 2 345 CTG ATG T Weak ATG 3 396 CCC ATG A Weak ATG 4 489 CTG ATG A Weak Hu-K4 A. Munck et al. 1722 FEBS Journal 272 (2005) 1718–1726 ª 2005 FEBS 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 ATG 1 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. A. Munck et al. Hu-K4 FEBS Journal 272 (2005) 1718–1726 ª 2005 FEBS 1723 hybridized using a [ 32 P]-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: NH 2 -LDTSADSVG NACRLL-COOH (coupled to keyhole limpet haemocyanin using glutaraldehyde) and NH 2 -CTWPRFYDTRYNQETP- CONH 2 (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. Exon Length G ⁄ C ATG Position Kozak consensus A GCC ATG G G Context Peptide 1 > 267 70% 44000 44013 CCA ATG A CGC ATG C Weak Weak >54aa 9aa 1¢ 44 68% – 2 > 53 20% 55046 TAT ATG T Weak 3 aa 2 ⁄ 4 55067 TCA ATG C Weak 58 aa 3 33 39% 60929 60933 60939 GTA ATG C TGC ATG T TCC ATG G G Adequate Weak Adequate 13 aa 33 aa 31 aa 3¢ 76 49% – 4¢ 56 61% 61041 GGA ATG T Adequate 1 aa 4¢ 61066 GCC ATG T Adequate 24 aa 4 214 73% – 5¢ 35 54% – ADB C 105 kDa 15 50 35 75 105 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 ). Hu-K4 A. Munck et al. 1724 FEBS Journal 272 (2005) 1718–1726 ª 2005 FEBS 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 indicated 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 NaH 2 PO 4 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. 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