A novel serine proteinase-like sequence from human brain

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Biochi~ic~a

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et Biophysica A~ta Biochimica et Biophysica Acta 1218 (1994) 225-228

Short Sequence-Paper

A novel serine proteinase-like sequence from human brain Melitta Dihanich *, Martin Spiess Department of Biochemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland (Received 20 December 1993)

Abstract

From Alzheimer's visual cortex mRNA, a novel cDNA sequence with homology to the carboxy-terminal two-thirds of known serine proteinases was isolated (43% identity with human trypsinogen II). The corresponding mRNA has a size of ~ 8 kb and is expressed in low amounts in all human tissues tested.

Key words: Serine proteinase; Precursor; Alzheimer's disease; Brain; (Human) Serine proteinases (EC 3.4.21) form a large, ubiquitous family of proteolytic enzymes involved in many important physiological and pathogenic processes. The nervous system has been shown to express serine proteinases, such as the two plasminogen activators and prothrombin [1-4], as well as several serine proteinase inhibitors [5-9]. Serine proteinases and inhibitors have been proposed to be involved in neuro-degenerative disorders such as Alzheimer's disease [10-14]. To clone novel serine proteinases of the brain, we employed a polymerase chain reaction (PCR) approach using oligonucleotide primers derived from chymotrypsin ( T T G A T C T A G A C C A G G G C T C T G A C G A G G A G A and C A G G G A A T T C G G A C C G C C A G A G T CGCCC corresponding to amino acids 90-96 with an additional XbaI site and to residues 211-216, the active site motif G D S G G P , with an added EcoRI site, respectively). Poly(A)+-selected m R N A [15] from human visual cortex Alzheimer's brain was reverse transcribed using an oligo(dT) primer. The resulting cDNA was used as a template for PCR. Amplified fragments were isolated from agarose gels and ligated into the vector pGEM3 for sequencing. Among a total of ~ 100 isolated PCR fragments only one, named A C O l l , contained a serine proteinase-like sequence. Using A C O l l

* Corresponding author. Fax: + 41 61 2672148. The sequence data reported in this paper have been submitted to the E M B L / G e n B a n k Data Libraries under the accession number X75363. 0167-4781/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 7 8 1 ( 9 3 ) 0 0 0 1 9 - Y

as a hybridization probe, three cDNAs could be isolated from a human Alzheimer's visual cortex cDNA library. All of them contained inserts of ~ 6 kb which overlapped with A C O l l only at the 3' end and yielded the 3' end of the coding sequence. Cloning the 5' sequence using R A C E (rapid amplification of cDNA ends) [16] was unsuccessful. In an attempt to clone the 5' end, we isolated 12 hybridizing clones from a genomic EMBL3 A-library with inserts between 15 and 20 kb. Upon subcloning into plasmid vectors large portions of insert sequence were lost even in a recA strain and using a low copy number plasmid. None of the recovered sequences extended A C O l l towards the 5' end, indicating that the upstream sequence is toxic for the host bacteria. The sequence of the PCR product A C O l l (nucleotides 1-1198) and of the 5' end of the cDNA clone A1 (nucleotides 916-1414) is shown in Fig. 1A and was named ACO. The deduced protein sequence is homologous to the carboxy-terminal two-thirds of serine proteinases. It shares 43% identical residues with type II human pancreatic trypsinogen and somewhat less with other serine proteinases (Fig. 1B). This is comparable to the homology between catalytic domains of other serine proteinases. However, ACO contains two sequence insertions which interrupt the homologous regions (underlined in Fig. 1A). An in frame insertion ot 49 codons (with respect to trypsinogen) is present following the conserved G W G motif (Fig. 1A, nucleotides 222-371, underlined) close to an intron position in several serine proteinases (e.g., kallikrein and chymotrypsinogen). This insertion could represent either an

M. Dihanich, M. Spiess / Biochimica et Biophysica Acta 1218 (1994) 225-228

226

unspliced intron (there are potential 5' and 3' splice sites) or could encode an additional peptide loop. PCR amplification of an ACO segment encompassing this region using cDNAs derived from different tissues and cell lines (see below, Fig. 3) yielded exclusively a fragment corresponding to the sequence including this insertion. A second insertion of 673 nucleotides interrupts the open reading frame immediately preceding the conserved active site sequence GDSGGP. It also coincides

with an intron position for example in the genes ot trypsinogen, chymotrypsinogen, and kallikrein. Splicing of this insertion would be required for the ACO sequence to encode an active serine proteinase. The ends of this inserted sequence conform with the consensus sequences for 5' and 3' splice junctions. However, PCR amplification of human frontal cortex cDNA using primers flanking this insertion (corresponding to nucleotides 424-443 and to the complement of nucleotides 1318-1336 in Fig. 1A) yielded the 913 bp

A AGGTGCGCTGGGAAAGCACAACCTGCGCAACGCGATGGCCCAGAGCAACTACGGACCACGTCTCGGGTcATTCCACACcCGCGCTACGAA R C A G K A Q P A Q R D G P E Q L R T T S R V I P H P R GCCAGCCACCGCAACGACATCATGTTGCTGCGCCTAGTCCAGCCCGcACGCCTGAACCCCCAGGTGCGCCCCGGGTGCTAC A S H R N D I M L L R L V Q P A R L N P Q V R P G C

Y

90 Y

E

CCACGCGTT P R V

GCCCCCACCCGGGAGGCCTGTGTGGTGTCTGGCTGGGGCTGGTGTCCCACAACGAGCCTGGGACCGCTGGGAGCCCCCGGTCACAAGGTG A P T R E A C V V S G W G W C P T T S L G P L G A P G H

K

V

CGTGAAAGGATGGAGCTGGATGCGAGGCCTCAAGGAATCCTATTGCTCCAGGGCTCTTGGGCGGAGGGGACAAGGGCCGGAATTTATGGA R E R M E L D A R P Q G I L L L Q G S W A E G T R A G I

Y

G

TCTGCTCCAAGTCCACTGTCTTCCCCAGTGAGTCTCC S A P S P L S S P V S L

P

270 360

CAGATACGTTGCATTGTGCCAACATCAGCATTATCTCGGACACATCTTGTGAC D T L H C A N I S I I S D T S C

AAGAGCTACCCAGGGCGCCTGACAAACACCATGGTGTGTGCAGGCGCGGAGGGCAGAGGCGCAGAATC K S Y P G R L T N T M V C A G A E G R G A E GGCCATCAGGCGGAAGAAGAGGGATGGGGACAGGTGTGGGAGTCCGGATGGGGTTGGATTTTCTTCGC

S

180

450 D

CTGTGAGGTCAGAGCCTAGAGG C E v r a *

540

TTTGGGC CAGAGAAGATGCTAG

630

~TTAGGCTTGGAGATGGTACGTAGGAAGAGAAGTTAGAATAGGGGTGAGGTTGGAGTTGGGGTTATAGGTGGGGATTGCGTTGTTTGAG

720

GTGGATAATGTGATAGTTAGTTTGAGATGGCATGGGTTGGGGTTGAGAATGGGAATGGTTTGGTTTGATTCTGGGTGGGAAATACGTCAG

810

GGTTGAATTGGGATGAGGTAGATTTTGTTTGGAATGCAGAAGACATGAAGATTGAGATTGGATTTTGAGATGGGCATGGGTTTGATTTGA

900

TTTTGAATGGTGAGCATGTGGGCTGAGTTGGATTTAAC

990

TTAGTACAGTTGCACTGGAGTTGCATGGGGGTGAGATTGGATATAGGTTGGG

TGAGTTGTATTGAGCTGTGTTGAATTGGGGTTGGGGTTGGGGTTGGGTTGGCTCTGTTTGGGATAAACTGGGCTGTATTGAGTTGAGTTG ~TTGGGGTTCCCTGGGATGGGGATGGATTGGGTTTGGGGTGAGATTGCAAATGGTGATTAGGATGAGGATGAATCCAGGAGGTTTCACT • i q e v

1080 1170 s

1

CAACCTGAGACC CCCTCTTTTCCC CACAGGTGACTCTGGGGGACCCCTGGTCTGTGGGGGCATCCTGCAGGGCATTGTGTCCTGGGGTGA n 1 r p p 1 f p t G D S G G P L V C G G I L Q G I V S W G

D

CGTCCCTTGTGACAACAC CACCAAGCCTGGTGTCTATACCAAAGTCTGCCACTACTTGGAGTGGATCAGGGAAAC V P C D N T T K P G V Y T K V C H Y L E W I R E ACTATTCTAGCCTATCTCCTGTGCCCCTGACTGAGCAGAAGCCCCCACAGCTGGCCAGCAGCCC

T

1260

CATGAAGAGGAACTG M K R N *

1350

1414

B ACO: Tryp: Kal:

...RCA G K A Q P A Q R D G P E Q L R T T S R VI PHPRYEASH RNDIMLLRLVQPAR LNPQVRPGCYPRVAPTR EACVVSGWGWC... ...VRL G E H N I E V L E G N E Q F I N A A K II R H P K Y N S R T L DNDILLIKLSSPAV INSRVSAISLPTAPPA AGTESLISGWG ...LFD D E N T A Q F V H V S E S F P H P G F N M S L L E N H T R QADEDYSHDLMLLRLTEPADTITDAVKVVELPTQEPE VGSTCLASGWG FIX: ...VVA-GEHNIEE T E H T E Q K R N V I R II P H H N Y N A A I N K Y N H D I A L L E L D E P L V LNSYVTPICIADKEYTNI FLKFGS GYVSGWG C h y m : ...VVA G E F D Q G S D E E N I Q V L K I A K,VF KNPK FSILTVNNDITLLKLATPAR FSQTVSAVCLPSADDDFP AGTLCATTGWG Throm:...VRI G K H S R T R , Y E R N I E K I S M L E K IY I H P R Y N W R E N L D R D I A L M K L K ~ V A FSDYIHPVCLPDRETAAS,LLQAGYKGRVTGWG

ACO: Tryp: Kal: FIX: Chym: Throm:

ACO: Tryp: Kal:

FIX: Chym: Throm:

(40 aa)...GIYGSAPSPLSSP V S L P D T L H C A N I S I I S D T S C D K S Y P G R L T N T ~ C A GA EG RGAESCE£6~)BGGPLVC NTLSSG ADYPDELQCLDAPVLSQAECEASYPGKITNNMFCV GFL EG G K D S C Q , 6 ~ S C ~ W SIEPEN-FSFPDDLQCVDLKILPNDECEKAHVQKVTDFMLCV GHL EG GKDTCV-GDSGGPLMC RVFHK G R S A L V L Q Y L R V P L V D R A T C L R S T K F T I Y N N ~ C A GFH EG GRDSCQ ~ S C ~ H V KTKYN,ANKTPDKLQQAALPLLSNAECKKSWGRRITDVMICA GAS GVSS(3M,GDSGGPLVC NLKETWTANVG KGQPSVLQVVNLPIVERPVCKDSTRIRITDNMFCA,GYKPDEGKRG DACE GDSGGPFVMK,

GG ILQGIVSWGDVPCDNTTKPGVTKVCHYLEWIRETMKRN* SNG ELQGIVSWGYG CAQKNRPGVTKVYNYVDWIKDTIAANS* DG VLQGVTSWGYVPCGTPNKPSVVR%rLSYVKWIEDTIAENS * TEVEGTSFLTGIISWGEE CAMKGKYGITKVSRYVNWIKEKTKLT* QKDGAWTLVGIVSWGSDTCSTSS PGVARVTKLIPWVQKILAAN* SPFNNRWYQMGIVSWGEG CDRDGKYGFTHVFRLKKWIQKVIDQFGE*

I~IhACO 100% 43% 37% 37% 37% 33%

M k ) n l ~ m Tgq) 43% 100% 41% 45% 42% 38%

Fig. 1. Sequence of ACO. (A) D N A sequence and deduced protein sequence for the large open reading frames. Insertion sequences without homology to known serine proteinases are underlined. Protein sequence within the second insertion are shown in lower case letters, and the active site motive is double-underlined. Stop codons are indicated as asterisks. (B) The deduced protein sequence of A C O without the intervening insertion of nucleotides 526-1198 (indicated by zx ) was aligned with the protein sequences of other serine proteinases: Tryp, h u m a n pancreatic trypsinogen II; Kal, h u m a n glandular kallikrein; FIX, h u m a n factor IX; Chym, h u m a n pancreatic chymotrypsinogen; Throm, h u m a n prothrombin. Residues conserved among all six sequences are shown in bold face, known intron positions by dots.

M. Dihanich, M. Spiess / Biochimica et Biophysica Acta 1218 (1994) 225-228

1

EBSt

23

4

227

8

567

910

kb --12 --

--

AC011 5

1

Fig. 2. Genomic Southern analysis. Genomic DNA from SK-N-SH neuroblastoma cells [18] was digested with restriction enzymes, size fractionated on an agarose gel, blotted to nitrocellulose, and hybridized with 32p-labeled A C O l l DNA. Restriction enzymes: E, EcoRI; B, BamHl; St, StyI.

unspliced fragment; a product corresponding to the spliced mRNA could not be detected (data not shown). In a Southern blot analysis of human genomic DNA digested with three different restriction enzymes (Fig. 2), ACO hybridized to single fragments indicating that it is encoded by a single gene. Expression of ACO mRNA, as tested by reverse transcription and quantitative PCR using specific ACO primers (Fig. 3), is low in the different tissues and cell lines analyzed: the best signal was obtained for human SK-N-SH neuroblastoma cells and human activated T-cells, which corresponded to approximately 10 .7/xg of standard A C O l l cDNA. Even lower signals were obtained with RNA from human visual cortex, hippocampus, liver, pancreas, kidney, spleen, prostate, testis, thyroid (in part shown in Fig. 3). Essentially no signal was detected with RNA from rat brain (Fig. 3, lane 8). There was no significant difference in ACO mRNA levels between Alzheimer's or normal brain (visual cortex and hippocampus), nor were there increased levels in different colon carcinoma ceils (SW620, S W l l l 6 , HT29, Colo205, CoSut; not shown). On Northern blots (Fig. 4) a band of ~ 8 kb was obtained with poly(A) ÷ enriched RNA from human brain and liver. The amount of signal seen with this method is in agreement with the PCR quantification. This large mRNA might be translated directly into a product lacking proteinase activity, similar to the a subunit of nerve growth factor, which is an inactive serine proteinase homolog [17]. Alternatively, it could represent an unspliced or partially spliced precursor RNA which might be further processed in certain cell

Fig. 3. PCR quantification of ACO mRNA in different tissues and cell lines. 20 ng of cDNA reverse transcribed from total RNA of different origin were used as templates for quantitative PCR [19] (1 min at 94°C, 2 min at 56°C, 3 min at 72°C, 30 cycles). The primers TCATCTAGATGTTGCTGCGCCTAGTCCA (nucleotides 424-443 in Fig. 1A) and ACAGAATTCAGATGTGTCCGAGATAATGC (the complement of nucleotides 1318-1336) allowed specific amplification of 331 bp ACO fragments. These were separated on a 1% agarose gel, blotted to nitrocellulose, and hybridized with a 32p_ labeled A C O l l probe. RNA was isolated from: activated human T lymphocytes MLR (mixed lymphocyte reaction; lane 1) and A i d (CD8-positive T-cell clone; lane 2); SK-N-SH human neuroblastoma cells (lane 3); IMR 322 human glioma cells (lane 4); human visual cortex (lane 5); human hippocampus (lane 6); human liver (lane 7); rat cortex (lane 8). 10 -4 ~g and 10 -6/xg A C O l l standard DNA(lanes 9 and 10). The same experiment performed with 20 cycles of PCR yielded less product in all lanes, but the same ratios (not shown).

types or under certain conditions to yield an active serine proteinase. The second hypothesis is supported by the fact that also prothrombin mRNA accumulates as a partially spliced precursor RNA in brain and neuronal cell lines [4]. We would like to thank Drs. J. Ulrich and M.J. Mihatsch (Institute of Pathology, Basel), B. Sordat (ISREC), G. Griffiths and P. de la Bonna (Basel Insti-

S

L

kb

~

--

10

--

8

--

5

m

1.9

q

Fig. 4. Northern blot analysis. Poly(A)+-selected RNA (10 ~g) ol SK-N-SH neuroblastoma cells (S) and liver (L) was size fractionated on a denaturing formaldehyde agarose gel, transferred to GeneScreen plus (DuPont), and probed with 32p-labeled ACOll.

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M. Dihanich, M. Spiess / Biochimica et Biophysica Acta 1218 (1994) 225-228

tute of Immunology), and D. Reddy (Hoffmann-La Roche) for tissue samples, cell lines and reagents. This study was supported by the Incentive Award of the Helmut Horten foundation.

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