Isolation and sequencing of a cDNA clone encoding the 85 kDa human lysosomal sialoglycoprotein (hLGP85) in human metastatic pancreas islet tumor cells

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Pages 604-611

Isolation and sequencing of a cDNA clone encoding the 85 kDa human lysosomal sialoglycoprotein (hLGP85) in human metastatic pancreas islet tumor cells Hideaki

Fujita, Takeshi

Yutaka Takata,* Akira Kono,* Yoshitaka Tanaka, Takahashi, Masaru Himeno and Keitaro Katol

Division of Physiological Chemistry, Faculty of Pharmaceutical Kyushu University, Higashi-ku, Fukuoka, Japan *National Kyushu Cancer Center, Minami-ku, Received

February

26,

Sciences,

Fukuoka, Japan

1992

SUMMARY:

A full length cDNA for a human lysosomal membrane sialoglycoprotein (hLGP85) was isolated as a probe of the cDNA of rat LGP85 (rLGP85) from the cDNA library prepared from total mRNA of QGP-1 NL cells, a human pancreatic islet tumor cell with a high metastatic activity. The deduced amino acid sequence shows that hLGP85 consists of 478 amino acid residues (MW. 54,289). The protein has 10 putative N-glycosylation sites and 2 hydrophobic regions at the NH*- and near the COOH-termini, respectively. Thus, both domains probably constitute putative transmembrane domains. It exhibits 86% and 79% sequence similarities in amino acids and nucleic acids to rat lysosomal membrane sialoglycoprotein (rLGP85), respectively. The protein contained the short cytoplasmic tail at the COOH-terminus which does not form the glycine-tyrosine sequence (GY motif), the so-called lysosomal 6 1992Academic Press,1°C. targetting signal.

Several lysosomal

membrane

glycoproteins

have been purified and isolated

their cDNA from various species (l-1 4). The cDNAs of lysosomal acid phosphatase and of two related families of lysosomal-associated membrane proteins (LAMPS), A and 6, have been characterized (l-7, 10, 11). These proteins have several common features in that they once span the lysosomal lipid bilayer through their hydrophobic COOH-terminus

domain which is located near their

and have a short cytoplasmic tail which contains the GY-motif

that is considered to be a lysosomal targetting signal (15, 16). These characteristics are well conserved in both LAMPS and acid phosphatase,

over

various species. Recently, Vega et al. and our group have isolated a lysosomal membrane glycoprotein belonging to another group of LAMPS, it twice spans the lysosomal lipid bilayer with an uncleavable signal peptide and hydrophobic domain located near the COOH-terminus (13, 14). As the protein does not 1To whom correspondence 0006-291X192 Copyright All rights

should be addressed.

$1.50

0 1992 by Academic Press. Inc. of reproduction in any form reserved.

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contain the GY-motif, it seems to be targeted to lysosomes in a manner different from that of LAMPS and acid phosphatase. As we wanted to determine various species,

we attempted

whether or not this protein is also conserved in to isolate the cDNA from human cell ( QGP-

1 NL), using the cDNA of rLGP85 as a probe (13). We report here the cloning and sequence of the cDNA related to rLGP85 from a human metastatic pancreatic

islet tumor cell line.

MATERIALS

AND METHODS

Establishment

of high

metastatic

cell line

(QGP-INL)

QGP-1 N was established from a human pancreatic islet cell carcinoma cell line (17, 18). The cells were injected into spleens of nude mice. After 40-60 days, the tumor nodule in the liver was isolated and a cell line was established. The new cell was injected into spleen of nude mice and the line was established from the nodule. These procedures were repeated 5 times and the metastatic cell was established as QGP-1 NL.

Construction

of cDNA library

from

QGP-1NL

RNAs were extracted from the confluent cultured QGP-1 NL cells by the method of Chirgwin et al. (19). Poly(A)+ RNAs were isolated by oligo-dT coupled resin (Oligotex-dT30, Takara, Kyoto). The first-strand cDNA was synthesized by using reverse transcriptase, a Oligo(dT)12-r8 as a primer according to the procedure established by Gubler and Hoffman (20). Secondstrand synthesis was carried out by the RNAase H procedure (19). Subsequent process to construct the hgtl 1 expression cDNA library followed the method of Young and Davis (21). The library consisted of 1.9x1 06 primary clones (QGP-1 NL cDNA library).

cDNA

Iibrary

screening

The 847 bp (nucleotides 645-1492 in rLGP85 cDNA) nucleotide fragments prepared by digestion of the cDNA of rLGP85 with Hincll endonuclease were labeled with [a-32P]CTP (>3000 CVmmol, Amersham) using the multiprime DNA labelling system (Amersham). Without further purification, the radioactive nucleotide fragments were used to screen QGP-1 NL cDNA library, using standard methods (22). To obtain cDNAs encoding hLGP85 from a QGP-1 NL cDNA library, the nucleotide fragment probes were used for clone isolation. Two positive clones isolated from approximately 4.2 x 1O5 phages, were termed hLGP-1 and hLGP-2 (insert length 2.3kb and 2.0kb), respectively.

DNA

sequencing

Restriction endonuclease fragments of cDNA of hLGP-1 (longer one) were subcloned into Ml 3mpl8,19 (23). The nucleotide sequences of the DNA were determined by dideoxynucleotide chain termination methods (24) using fluorescent dye-labeled oligonucleotides (Applied Biosystems’ Dye-primers) and single stranded M13DNA by an Applied Biosystems Model 373A DNA Sequencer with automated electrophoresis detection system.

Computer

analysis

of cDNA and protein

Nucleotide and protein sequences (Software Development Co., LTD.).

were analyzed using the GENETYX

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Preparation

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fractions

Rat liver lysosomal membranes were prepared according to the method of Ohsumi et al. (25). The confluent cultured QGP-1 NL cells were harvested and the cells were homogenized with 10 mM sodium phosphate buffer (pH 7.0). The resulting homogenates were centrifuged for 5 min at 650xg to remove nuclei and unbroken cells and the thus obtained supernatants were subjected to further centrifugation for 1 h at 105,OOOxg. The pellets served as the membrane fractions . Human liver was obtained at autopsy on a person found dead on the street and the membrane fractions were prepared as described above. Human placenta membrane fractions were kindly donated by Dr. Y. lkehara (Fukuoka University).

Western

b/o tting

SDS/PAGE in a 10% slab gel was carried out as described (26). After electrophoresis, proteins were transferred electrophoretically to nitrocellulose sheet as described by Towbin et al. (27). For immune visualization, the nitrocellulose sheet was first soaked in phosphate- buffered saline (10 mM sodium phosphate buffer, pH 7.5, 0.15 M NaCI) containing 0.05% Triton X-l 00, 2% bovine serum albumin, for 60 min, followed by incubation with 30 yg/ml of a rabbit monospecific IgG against rLGP85 for 60 min. The sheet was then soaked in ‘251-labeled protein A solution for 60 min. The excess second step 1251-labeled protein A was removed by three washes in the same above buffer containing 0.05% Triton X-l 00, then the sheet was exposed to Kodak X-OMAT film.

RESULTS

Isolation

AND

DISCUSSION

of cDNA

clones

A QGP-1 NL cDNA library constructed with hgtl 1 as a vector (21) and screened with the nucleotide fragments of two positive termed

prepared from the cDNA of rLGP85 led to isolation

clones from approximately

hLGP-1 and hLGP-2, respectively.

4.2 x105 phages.

The clones were

hLGP-1 with a longer cDNA insert

was named hLGP85 and subjected to DNA sequencing.

Sequence

analyses

of the cDNA

and structure

of hLGP85

The longer cDNA fragment (hLGP85) was subcloned into the plasmid vector pUC118 and analyzed by restriction mapping. Fig. 1 shows the complete nucleotide primary hLGP85 flanked region. typical

sequence

structure

determined

of hLGP85.

from the hLGP85 As shown

cDNA and the deduced

in Fig. 1, the cDNA fragment

of

contains an entire coding region of hLGP85 (nucleotides 252-1688) by 251 nucleotides of 5’- and 641 nucleotides of 3’- untranslated Although a poly(A) tail was not found, at position 2279 there is a polyadenylation signal (AATAAA). The hLGP85 cDNA potentially

encodes a 478 amino acid polypeptide, starting from the first initiation codon (ATG). An in frame TGA stop codon is located 15 nucleotides upstream from

606

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10 20 30 40 50 60 70 80 90 CRCGGCTtCCCGGCG*GGRAACCGAAACCGAGTcc~GcccGTcccTccGcGGccccA*ccGcccGGTG*cccGGGGccGcGcTcGcc 100 110 120 130 140 150 160 170 180 AGGCCGCGGAGCCAGAGCTGCGCGCACGAACCGTGCGCGGCGCCTCTGCG 190 200 210 220 230 240 250 260 270 GCGGCTCCTCCCTCCTTGCAGTTGGATCCCTGGCGGGTGCGGCCCGGCCCGGCCCG~~.~GCGGCGCACAG~TGGGCCGATGCTGCTTCT M,G R[ C C F 1

280 290 300 310 320 330 340 350 360 ACACGGCGGGGACGTTGTCCCTGCTCCTGCTGGTGACCAGCGTCACGCTGCTGGTGGCCCGGGT,:TTCCAG~GGCTGTAGACCAGAGTA k T A G T T. S 7, T. 7, L" T S V T L T, 'JAR V F Q K A" 0 Q s 370 380 390 400 410 420 430 440 TCGAGAAGFLAAATTGTGTTAAGGAATGGTACTGAGGCATGCCCCCTCTGCCTGTGTATACTCAGTTC*ATTTCT I E K K IVLRNGTEAFDSWEKPPLPVYTQFYF x 460 470 480 490 500 510 520 530 TCAATGTCACCRATCCAGAGGAGATCCTCAGAGGGGAGACCA FNVTNPEEI LRGETPRVEEVGPYTYRELRN # 550 560 570 580 590 600 610 620 AAGCAAATATTCAATTTGGAGATAATGGAACAACAATATCTGCTGTTAGC~C~GGCCTATG~TTTTG~CGAGACC~TCTGTTGGAG K A N I *PGDNGTTISAVSNKAYVFERDQSVG ii 640 650 660 670 680 690 700 710 ACCCT-TTGACTTAATTAGAACATTRAATATTCCTGTTCATCG 0 P K I D L I R T L N IP"LT"IEWSQ"HFLREII

730 740 750 760 770 AGGCCATGTTGAAAGCCTATCAGCAGAAGCTCTTTGTGACTCTTGTCCC EAMLKAYQnKLFVTHTVDELLWGYKDEILs

780

790

800

820 830 840 850 860 870 880 890 TTATCCATGTTTTCAGGCCCGATATCTCTCCCTATTTTGGCCTATTCTATGAG~TGGGACT~TGATGGAGACTATGTTTTTCT~ L I HVFRPDI s P Y F G LFYEKNGTNDGOYVFL

450

540

630

720

810

900

x 910 920 930 940 950 960 970 980 990 CTGGAGAAGACAGTTACCTTAACTTTTAC~TTGGM T G E 0 SYLNFTKIVEWN GKTSLDWWITDKCN x 1000 1010 1020 1030 1040 1050 1060 1070 1080 TGATTRATGGAACAGATGGAGATTCTTTTCACCCACTRATCTTTTGCAGGTCAG MINGTDGO SFHPLITKDEVLYVFPSOFCRS x 1090 1100 1110 1120 1130 1140 1150 1160 1170 TGTATATTACTTTCAGTGACTATGAGAGTGTACAGGGACTGCC~GCCTTTCGGTAT-GTTCCTGCAG-TATTAGCC~TACGTCAG " Y ITFSDYCI S"aGLPAFRYK"PAEI L A N T S Y llB0 1190 1200 1210 1220 1230 1240 1250 1260 ACAATGCCGGCTTCTGTATACCTGAGGGARACTGCCTGGGTGGTGCACCCATCATTA DNAGFCIP E G N C L G S G ‘v' L N " S ICKNGAPII li 1270 1280 1290 1300 1310 ~ 1320 1330 1340 1350 TGTCTTTCCCACACTTTTACCAAGCAGATGAGATGTCATTTG M s F P HFYQADERFVSA IEGMHPNQEDHETF

1360 1310 1380 1390 1400 1410 1420 TGGACATTAATCCTTTGACTGGAATAATCCTAAAAGCAGCC~GAGGTTCCA~TC~CATTTA~GTC~ " D I NPLTGII LKAAKRFQINIYVKKLDDF"

1430 1440 TTAGATGACTTTGTTG

1450 1460 1470 1480 1490 1500 1510 1520 1530 AAACGGGAGACATTAGRACCATGGTTTTCCCAGTGATGTACCTCARTGAGA~~GTGTTCACATTGATAAAGAGACGGCGAGTCGACTGAAGT E T G D I RTMVFPVMYLNE s " H I DKETASRLK Y 1540 1550 1560 1570 '1580 1590 1600 1610 1620 CTATGATTAACACTACTTTGATCATCACCARCATACCCTACCCTACATCATCATGGCGCTGGGTGTGTTCTTTGGTTTGGTTTTTACCTGGCTTG S M I NIT T I. I T T N T P Y I I M A I, G" p F G L" F T w r,~ # 1630 1640 1650 1660 1670 1680 1690 1700 1710 CATGC-GGA~AGGGATCCATGGATGA~GG~~A~CGGAT~~~A~CACC~~T~A~~~~~CCT~ACAT~~~~~TT~~TTGGTG~G =KGQGSMDEGTAD ERAPLIRT' 1720 1730 1740 1750 1760 1170 1780 1790 1800 AAACTGTGTGAGCTGTCCTGACCTGGACGATGACGTGGGGRAACCCTCCACCTCCTTGCAGGCTTGTTGCCTGTTGAAAGAAGGAAAAAG 1810 1820 1830 1840 1850 1860 1870 1880 1890 A~AC~GCGCTGGCAAGTGATAGGAACATTCTGGCCAGAGG~~AGAGGTT-GAG~AGG~TGACATGG~TGGCCATT~GCTTTAT-TCATGTG 1900 1910 1920 1930 1940 1950 1960 1970 GGCTCTGAAATTGTTCTTTTATGTGT~TAGC~GTATTTAAT~ACCCTTGTATAGT~TTTTGTTGTTGTTGGGTG~TGGTAGCTCCAG 1990 2000 2010 2020 2030 7040 905" sn+Tn

1980 9"7r-

2080 2090 2100 2110 2120 2130 2140 2150 2160 ACTTTGTGCTCAAAATGCGTATATACCATTTTATGTTGTATTCCTCCATTTCACTTGCARAACAGAAGTAAATAAGAGTTCGGGACCCAG 2170 2180 2190 2200 2210 2220 2230 2240 2250 GGTAAAATGGTAG~TTCAT~~~TATTCAAAT~ATTCA~TGC~~~TGATTTCT-~~ATATTAC~TTTTATGCTGAT~TTCAGTTCAT~TT 2260 2210 2280 2290 2300 2310 2320 CTTCCAGGM,AACTCAGTCTTCCAACTGC BBTBBBRTACTGGGGTAGGATCAAATGGGGAAAGGGGGGGGGGGGGGGCC

fro. 7. Nucleotide sequences of cloned cDNA and deduced amino acid sequence of hLGP85. The deduced amino acid sequence is shown below the nucleotide sequence. Nucleotides are numbered above the lines. Shadowed boxes indicate a stretch of hydrophobic amino acids, possible transmembrane domains, in the Nf-fs- and at near the COOH- termini. Asparagine residues (#) represent potential N-linked glycosylation sites. An underline in the noncoding region indicates the polyadenylation signal. The stop codons limiting the open reading frame are indicated by asterisk. Broken underline indicates in frame stop codon and the arrowhead the likely posttranslational cleavage site. 607

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4

fit. 2. SDS/PAGE of membrane fractions prepared from rat liver, human liver, QGP-lNL, and human placenta. Each membrane fraction was subjected to SDS/PAGE followed by immunoblot analysis, using specific anti-rLGP85lgG. Lane 1, rat liver lysosomal membranes ; Lane 2, membrane fractions from human liver ; Lane 3, membrane fractions from QGP-1 NL; Lane 4, membrane fractions from human placenta.

the initiator ATG. Since we did not determine

the NH*-terminal

sequence

of

hLGP85, we do not know whether it started at initiator methionine or at the second glycine. However, as initiator methionine in rLGP85 was cleaved off, in the case of hLGP85 the second glycine is probably an NH2-terminal acid (13) as suggested

by Sherman et al. (28). Thus, it is presumed

putative signal peptide cannot be cleaved during biosynthesis as a potential

membrane

spanning

domain

starting with glycine after initiation methionine

of hLGP85.

amino that the

and it remains The polypeptide

as its amino terminus would be

477 amino acids long and have a molecular mass of 54,158. The entire primary structure contains 10 potential N-glycosylation sites (Asn-X-Thr/Ser). Since Western blot analysis showed that the molecular mass was 85K, glycosylation of some of these asparagine residues could account for the difference

between

the molecular

mass calculated

from the amino

acid

composition (54,158 Da) and that estimated by SDS/PAGE for the mature protein (85 kDa). Fig. 2 shows Western blot analyses of proteins from rat liver lysosomal membranes, human liver membrane fractions, QGP-1 NL membrane fractions, and human placenta membrane fractions, respectively. rLGP85 and rLGP85 like protein in human placenta showed the same mobility and the proteins from QGP-1 NL and human liver moved the same distance on SDS/PAGE. 608

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When mobilities of LGP85 in rat liver and human placenta were compared

to

those of the proteins in QGP-1 NL and human liver, the latter two migrated slightly faster. Since hLGP85 and rLGP85 have 10 and 11 potential glycosylation sites, respectively, we conclude from the Western blot analysis that the N-glycosylation sites used for glycosylation in these proteins are not much different in number because

proteins from rat liver lysosomal

membranes, human liver, human placenta and QGP-1 NL showed a similar migration in SDS/PAGE. As Dennis et al. (29, 30) suggested that 81,6 branching of N-linked oligosaccharides (LAMP-l

on lysosomal membrane

and P2B are the same protein) in MDAY-D2

protein

results in a molecular

weight increase of the protein and are directly associated with a high metastasis, we expected the protein from QGP-1 NL ( which has a high metastasis) to be larger than the protein from cells such as human liver and placenta.

However, such a molecular weight increase was not observed.

Comparison

of hLGP85

with rLGP85

and

CD36

The cDNAs of acid phosphatase and of two related families of lysosomal membrane proteins such as LAMP A and B were characterized (l-7, 10, 11) and they proved to be well conserved also

anchored

over the different species. These proteins are

in the lysosomal

lipid bilayer

with a hydrophobic

domain

localized near the COOH-terminus and a short cytoplasmic tail followed the hydrophobic domain. The cytoplasmic tail forms a GY-motif that is considered to be an important

signal for lysosome

targetting

(15, 16, 31).

CD63 which is

originally described as a blood platelet activation marker was cloned as a lysosomal membrane protein. Although the protein has four hydrophobic domains

and thus spans lysosomal

short cytoplasmic motif is formed

lipid bilayers four times, the protein has a

tail after the COOH-hydrophobic

domains

in which the GY-

(12). The protein seems to belong to group mentioned (13, 14) seems

to belong

to another

above.

However,

the LGP85

new group

lysosomal lysosomal

membrane proteins because the protein was anchored in the lipid bilayer with near the NH,- and COOH-terminal hydrophobic

domains and LGP85 has a short cytoplasmic tail after the COOH-terminal hydrophobic domain, in which GY-motif was not formed. Since lysosomal membrane proteins are well conserved in various species, we were interested to see whether rLGP85 was also so well conserved. We attempted rLGP85.

to isolate the clone from human cells hybridizable The clone hybridized

to the cDNA of

to the cDNA of rLGP85 was isolated

as a

hLGP85, both proteins constituted the same number of amino acid residues and the two hydrophobic domains in hLGP85 are located the same positions in LGP85. In the case of N-glycosylation sites, all the sites in rLGP85 are conserved in hLGP85 except one. The potential N-glycosylation sites in hLGP85 is only one less than those of rLGP85 because of conversion from 609

in

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MGRCCFYTAGTLSLLLLVTSVTLLVARVFQKAVDQSIEKKIVL *.f**h*******X*k****~~~~~~~~~~~~*~~.~~~ MARCCFYTAGTLSLLLLVTSVTLLVARVFQKAVDQTIEKNMVL

rLGP85

RESEARCH

COMMUNICATIONS

60

. . KVFDSWEKPPLPV

hLGP85

TISAVSNKAYVFE

rLGP85

TISAVTNKAYIFE

120 hLGP85

RDQSVGDPKIDLIRTLNIPVLTVIEWSQVHFLREIIEAMLKAYQQKLFVTH?VDELLWGY * ******e.***Xf *** *** * * ***************

rLGP85

~GDPT'JDLIRT;NIPLLTWEMAQQPFLREIIEAMLKAYQQTLFVTHTVP.ELLWGY

hLGP85

KDEILSLIHVFRPDISPYFGLFYE *** **********-** KDEVLSLVHIFRPDVSPNFGLFYE

rLGP85

*******

180

G NDGDYVFLTGEDSY ***********A**

******.**

240 KIVZWNGKTSLDWW

NDGEYVFLTGEDN"

B

hLGP85

DGDSFHPLITKDEVLYVFPSDFCRSVYITFSDYESVUGLPAFRYKVPAE *********.***.**.****************..*.r.************

rLGP85

DGDSFHPLISKDETLYIFPSDFCRSVYIT~SSFENVEGL?AFRYKVPAE

hLGP85

******

300

ICKNGAPIIMSFPHFYQADERFVSAIEGMHPNQ **h***********hf****.~~~~*.~~.~~.

rLGP85

ENAGFCIPEGNCMDAGV

hLGP85

EDHETFVDINPLTGIILKAAKRFQINIYVKKLDDFVETGDIRTMVFPVMY X.**.*********X**..*******.*fX*******k~.~~~~~~~~~~

rLGP85

EEHESFVDINPLTGIILRGAKRFQINTYVKKLDDFVETGNIRT~FPVMY

hLGP85

TASRLKSM x******.

rLGP85

TASQLKSV B

NT x*

360

ICKNGAPIIMSFPHFYQADEKFVSAIKGMRPNK

420

LIITNIPYIIMALGVFFGLVFTWLACKGQGSMDEGTADERPLIRT **.****h***********.~~~~~~.~~~~ LIVTNIPYIIMALGVFFGLIFTWLACRGQGSTDEGTADFRAPLIRT

I*************

478

amino acid sequences of hLGP85 and rLGP85. Amino acids are numbered on the right. Shadowed boxes indicate potential asparaginelinked glycosylation sites. Asterisks indicate identical residues and single dots indicate conserved residues.

Fig. 3. Aligned

asparagine together, gene other

at position we conclude

122 to aspartic

and that LGP85 may be conserved lysosomal

acid, as shown

that hLGP85 and rLGP85 originated

membrane

in Fig. 3. Taken from the same

over various species as are three

glycoproteins

(LAMP

A,

LAMP

8,

Acid

phosphatase).

This study ACKNOWLEDGMENTS: We thank M. Ohara for helpful comments. was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.

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