lnt J Clin Lab Res 21:256-263, 1992 9 Springer-Verlag 1992

Molecular cloning and sequence analysis of the cDNA encoding human apolipoprotein H (flz-Glycoprotein I) * Joseph R. Day 1, Patrick J. O ' H a r a and Philippe Arnaud 3

2, Francis

J. Grant 2, Catherine Lofton-Day 2, Mary N. Berkaw 3, Phillip Werner 3,

1 Department of Medicine, University of Washington, XD-48, 2121 North 35th Street, Seattle, WA 98103, USA 2 Zymogenetics Corporation, Seattle, WA 98105, USA 3 Department of Microbiology and Immunology, Medical University of South Carolina. Charleston, SC 29425, USA

Summary. Apolipoprotein H, also known as fl-2-glycoprotein I, was purified from human serum, and antiserum produced to denatured apolipoprotein H detected a c D N A clone from a 2 gtl 1 library derived from human liver. This c D N A coded for the complete sequence of the mature protein. The c D N A insert, along with a polymerase chain reaction product which extended the 5' end of the message, were subcloned and both strands were sequenced. The apolipoprotein H precursor was found to code for 345 amino acids, 326 of which appear in the mature protein. The deduced amino acid sequence of human apolipoprotein H differs from its rat homologue by the presence of a 48-amino acid stretch which is absent from the rat protein. The remainder of the proteins share a greater than 80% similarity. The amino acid sequence of apolipoprotein H consists largely of repeated units approximately 60 amino acids in length. These repeats are comparable to "sushi structures" found in a large number of diverse proteins, including complement components, receptors and regulators of complement activation, serum proteins, membrane-associated adhesion proteins, and other structural and catalytic proteins. Apolipoprotein H was shown to be transcribed by human hepatoma cell lines Hep 3B and Hep G2, and rat liver by detection o f m R N A using northern blot analysis. Key words: Apolipoprotein H - Molecular cloning - Se9quence analysis

tent of 19%, and a normal plasma concentration of approximately 0.2 mg/ml. The protein was first isolated by Schultze et al. [65]. It was renamed Apo H [491 when it was shown to be an activator of lipoprotein lipase. Apo H has been shown to be a structural component of chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL) [59]. Although the precise function of Apo H is not known, it is thought to be involved in lipid metabolism for a number o f reasons. Human Apo H activates lipoprotein lipase [49], 35% of Apo H in man is associated with lipoproteins [59], and it has been shown to enhance triglyceride removal in rats [76]. Apo H may also be involved in coagulation since it inhibits the prothrombinase activity of human platelets [55]. Apo H protein consists of a single polypeptide chain containing 326 amino acid residues [46]. Apo H has five equivalent segments, each o f about 60 residues in length, which contain cysteines with similar disulfide-bonding patterns. This repeating 60-amino acid unit has been shown to be a c o m m o n structural element in a large number o f diverse proteins [27]. We have recently isolated and cloned a c D N A coding for human Apo H. In this article, we present the sequence of the full-length human c D N A and compare it with the reported rat c D N A sequence. In addition, the results of screening several tissues and cell lines for the presence of Apo H message are reported. Finally, the repeated motif o f A p o H is compared with similar motifs found in several plasma and structural proteins.

Introduction Materials and methods Apolipoprotein H (Apo H), originally called fl-2-glycoprotein I, is a plasma glycoprotein which has an approximate molecular weight of 50 kDa, a carbohydrate con* The nucleotide sequence data were deposited in the GenBank Nucleotide Sequence Database, accession number M62839 (April 4, 1991) Offprint requests to: J.R. Day

Purification of Apo H. Apo H was purified from normal human

serum using a two-step purification procedure [16]. Forty milliliters of human serum were dialyzed against 0.02 M Sodium Phosphate, 0.02% sodium azide, pH 7.0 (equilibration buffer), and then loaded onto a 2.5 x 100cm Affigel Blue column (Bio-Rad, Richmond, Calif.) as previously described [22]. Bound Apo H was eluted using a 0-1.5 M sodium chloride gradient and detected by fused rocket immunoelectrophoresis. The second step of the purification utilized

J.R. Day et al.: Molecular cloning and sequence analysis of the cDNA encoding human apolipoprotein H preparative 5%-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pooled column fractions containing Apo H were boiled in non-reducing SDS-loading buffer, loaded onto gels, and detected by protein staining with Coomassie blue and western blotting using commercially available antisera (Behring Diagnostics, Somerville, N.J.). Sections of the acrylamide gel containing Apo H were excised and electroeluted. The resulting purified Apo H was reduced and denatured by boiling in SDS-loading buffer containing dithiothreitol, then used to immunize a rabbit. The resulting antiserum was used for immunoscreening of the liver cDNA library.

Immunoscreening ofcDNA library. Plaques of a human liver cDNA library in 2 gtl 1 [28] were screened with the monospeciflc antiserum raised to denatured and reduced Apo H. The 2 g t l l library was grown on a lawn of Escherichia coli, and lac Z-directed gene expression was induced by placing an isopropyl-fl-D-thiogalacto-pyranoside-soaked nitrocellulose membrane (Schleicher and Schuell, Keene, N.H.) on the agar plate. After incubation at 37 ~ for 4 - 6 h, the membrane was removed and blocked with 5% dry milk in TRISbuffered saline (TBS). The membrane was then incubated with rabbit antihuman Apo H, washed with TBS, and incubated with goat antirabbit IgG horse radish peroxidase (HRP)-conjugated antibody (Cappel, Downington, Pa.). After washing, the positive clones were visualized with an HRP color development assay (Bio-Rad).

DNA subcloning and sequencing. The Apo H cDNA insert was subcloned into the EcoRI sites of pUC19 (Boehringer Mannheim, Indianapolis, Ind.) and pBluescript KSII (Stratagene, LaJolla, Calif.). The cDNA insert encoding Apo H was sequenced by the dideoxy chain termination method [63] using 3Ssulfur-dATP from New England Nuclear (Boston, Mass.) [6]. The reactions were catalyzed by modified T7 DNA polymerase (Pharmacia, Piscataway, N.J.) [70], and were primed with a universal primer, reverse primer, or with oligonucleotides complcmentary to the DNA region of interest. Double-stranded templates were denatured with sodium hydroxide [11] prior to hybridizing with a sequencing oligonucleotide. Oligonucleotides were synthesized on an Applied Biosystems (Foster City, Calif.) model 380 A DNA synthesizer. All sequences were determined on both strands. Rapid amplification ofcDNA ends. The 5' region of Apo H mRNA was obtained using rapid amplification ofcDNA ends (RACE) [20]. Briefly, first-strand eDNA was reverse transcribed from 1 lag Hep G2 poly (A) + RNA utilizing an Apo H-specific oligonucleotide primer, spin filtered using a Centricon-100 filter, then tailed with I mM dATP using 5 units of terminal transferase. The resulting product was diluted to 500 ~tl and a 5-~tl aliquot was used for amplification. The cDNA pool, (dT)lT-adaptor (40 pmol), specific primer (20 pmol), and 60 lal 1 x polymerase chain reaction buffer (Perkin-Elmer-Cetus, Norwalk, Conn.) were denatured (95 ~ 5 min) and cooled to 75 ~ Taq DNA (2.5 units) polymerase (AmpliTaq, Perkin-Elmer-Cetus) and 4 lal 5 mM of each deoxy-triphosphate nucleotide (Pharmacia) were added and the reaction mix overlaid with paraffin oil. Thirty-five cycles of amplification were carried out in a DNA thermal cycler (Perkin-Elmer-Cetus) using a step program (94~ 1 min; 45 ~ 2 min; 72~ 4 rain), followed by a 10-rain final extension at 72 ~

Cell culture and enzyme-linked immunosorbent assay. Human Hep 3B and Hep G2 cell lines (American Type Culture Collection) were grown in minimum essential medium (Hazelton Research Products, Denver, Pa.) supplemented with 10% fetal calf serum, 100 laM sodium pyruvate, 25 mM Hepes, 1% penicillin/streptomycin until confluent. Aliquots of protein secreted by Hep 3B and Hep G2 were analyzed by enzyme-linked immunoabsorbent assay (ELISA). Secreted Apo H was detected using a 1:2000 dilution of rabbit anti-Apo H antiserum (Behring Diagnostics) as the primary antibody, a 1:4000 dilution of goat anti-Apo H (Atlantic Antibodies, Scarborough, Me.) as secondary, antibody followed by HRP-labeled rabbit antigoat antibody at a dilution of 1:750 (Cappel).

257

RNA isolation and northern analysis. RNA was prepared from Hep 3B, Hep G2, Jurkat (human leukemic T cell), THP-I (human leukemic monocyte), and several rat tissues by a single-step total RNA isolation method of acid/guanidinium thiocyanate/phenol chloroform extraction [12]. Total RNA (5-20 lag) was denatured with formamide and formaldehyde and analyzed by electrophoresis on a 1.20 (w/v) agarose gel containing formaldehyde. The gels were dried and hybridized directly [73] with an Apo H probe labeled by random priming using a commercially available kit (BoehringerMannheim, Indianapolis, Ind.). Hybridization was conducted overnight at 65 ~ in a shaking water bath. The 32phosphorus-labeled probe, in 0.2 x TE containing salmon sperm DNA, was boiled for 5 min then added to a heat-sealable bag containing 4 x SSC, 0.5% dry milk, and 1% SDS for the hybridization step. Gels were then washed five times for 15 min at room temperature and three times at 60~ in an excess of 1 x SSC, 0.1% SDS solution. Gels were redried and exposed to XAR X-ray film (Eastman Kodak, Rochester, N.Y.) for 2-24 h.

Sequence analysis. The DNA sequences representing Apo H mRNA were analyzed on Sun SPARC Station I and Compaq 386 computers using Intelligenetics (Mountain View, Calif.) software and homology search programs [2, 8, 45, 57]. The sequences were compared with the GenBank [7] and EMBL [24] nucleotide sequence databanks, the PIR protein identification resource protein databank [21] and the SWISSPROT protein sequence database (A. Bairoch, Department de Biochemie Medieale, Centre Medical Universitaire, 1211 Geneva 4, Switzerland). The significance of matches with database sequences was assessed by the RDF algorithm of Lipman and Pearson [45]. Oligonucleotide primers were designed with the aid of software distributed by Scientific Computing Services (Seattle, Wash.).

Results Cloning o f Apo H A p o H is a p l a s m a p r o t e i n w h i c h shares several p r o p e r ties with p l a s m a a p o l i p o p r o t e i n s . T h e m a j o r sites o f synthesis o f o t h e r a p o l i p o p r o t e i n s are the liver a n d / o r intestines [75], t h e r e f o r e it seemed likely t h a t A p o H w o u l d be synthesized in the liver. W e s t e r n b l o t analysis o f unred u c e d p r o t e i n s secreted b y H e p 3B cells revealed the presence o f A p o H ( d a t a n o t shown). H e p 3B is a well-characterized cell line t h a t retains the a b i l i t y to synthesize a n d secrete m o s t o f the p r o t e i n s p r o d u c e d by n o r m a l liver p a r e n c h y m a l cells [34], a n d m o s t o f the m a j o r p l a s m a a p o l i p o p r o t e i n s [77]. A h u m a n liver c D N A l i b r a r y was screened with two different c o m m e r c i a l a n t i s e r a m a d e to native A p o H, b u t the clones identified w i t h these antise r a p r o v e d to be u n r e l a t e d to the p r o t e i n sequence o f A p o H as r e p o r t e d b y L o z i e r et al. [46]. W e s t e r n b l o t analysis revealed that, a l t h o u g h these a n t i s e r a r e c o g n i z e d the native f o r m o f the p r o t e i n , t h e y failed to recognize r e d u c e d a n d d e n a t u r e d A p o H [16]. T h e p r o t e i n was then purified f r o m h u m a n s e r u m a n d a n t i b o d i e s p r o d u c e d to r e d u c e d a n d d e n a t u r e d A p o H were used to i m m u n o s creen the s a m e library. O n e p o s i t i v e p l a q u e was selected a n d purified. T h e insert o f this p h a g e was excised with EcoRI, s u b c l o n e d into p U C 1 9 a n d s e q u e n c e d by the m e t h o d o f S a n g e r et al. [63] as m o d i f i e d b y C h e n a n d S e e b u r g [11]. T h e insert o f this p l a q u e was f o u n d to be 1116 nucleotides in length, b u t the 5' e n d d i d n o t c o n t a i n an initiating m e t h i o n i n e .

258

J.R. Da? et al.: Molecular cloning and sequence analysis of the c D N A encoding human apolipoprotein H

1 -19

GAAAACCACTTTGGTAGTGCCAGTGTGACTCATCCACAATGATTTCTCCAGTGCTCATCTTGTTCTCG MetIleSerProValLeuIleLeuPheSer

68 -I0

69 A G T T T T C T C T G C C A T G T T G C T A T T G C A G G A C G G A C C T G T C C C A A G C C A G A T G A T T T A C C A T T T T C C A C A -9 S e r P h e L e u C y s H i s V a l A l a I l e A l a G l y A r g T h r C y s P r o L y s P r o A s p A s p L e u P r o P h e S e r T h r

137 +14

138 G T G G T C C C G T T A A A A A C A T T C T A T G A G C C A G G A G A A G A G A T T A C G T A T T C C T G C A A G C C G G G C T A T G T G +15 V a l V a l P r o L e u L y s T h r P h e T y r G l u P r o G l y G l u G l u I l e T h r T y r S e r C y s L y s P r o G l y T y r V a l

206 +37

207 T C C C G A G G A G G G A T G A G A A A G T T T A T C T G C C C T C T C A C A G G A C T G T G G C C C A T C A A C A C T C T G A A A T G T +38 S e r A r g G l y G l y M e t A r g L y s P h e I l e C y s P r o L e u T h r G l y L e u T r p P r o I l e A s n T h r L e u L y s C y s

275 +60

276 A C A C C C A G A G T A T G T C C T T T T G C T G G A A T C T T A G A A A A T G G A G C C G T A C G C T A T A C G A C T T T T G A A T A T +61 T h r P r o A r g V a l C y s P r o P h e A l a G l y I l e L e u G l u A s n G l y A l a V a l A r g T y r T h r T h r P h e G l u T y r

344 +83

345 C C C A A C A C G A T C A G T T T T T C T T G T A A C A C T G G G T T T T A T C T G A A T G G C G C T G A T T C T G C C A A G T G C A C T +84 P r o A s n T h r I l e S e r P h e S e r C y s A s n T h r G l y P h e T y r L e u A s n G l y A l a A s p S e r A l a L y s C y s T h r

413 +106

414 G A G G A A G G A A A A T G G A G C C C G G A G C T T C C T G T C T G T G C T C C C A T C A T C T G C C C T C C A C C A T C C A T A C C T +107 G l u G l u G l y L y s T r p S e r P r o G l u L e u P r o V a l C y s A l a P r o I l e I l e C y s P r o P r o P r o S e r I l e P r o

482 +129

483 A C G T T T G C A A C A C T T C G T G T T T A T A A G C C A T C A G C T G G A A A C A A T T C C C T C T A T C G G G A C A C A G C A G T T +130 T h r P h e A l a T h r L e u A r g V a l T y r L y s P r o S e r A l a G l y A s n A s n S e r L e u T y r A r g A s p T h r A l a V a l

551 +152

552 T T T G A A T G T T T G C C A C A A C A T G C G A T G T T T G G A A A T G A T A C A A T T A C C T G C A C G A C A C A T G G A A A T T G G +153 P h e G l u C y s L e u P r o G l n H i s A l a M e t P h e G l y A s n A s p T h r I l e T h r C y s T h r T h r H i s G l y A s n T r p

620 +175

621 A C A A A A T T A C C A G A A T G C A G G G A A G T A A A A T G C C C A T T C C C A T C A A G A C C A G A C A A T G G A T T T G T G A A C +176 T h r L y s L e u P r o G l u C y s A r g G l u V a l L y s C y s P r o P h e P r o S e r A r g P r o A s p A s n G l y P h e V a l A s n

689 +198

690 T A T C C T G C A A A A C C A A C A C T T T A T T A C A A G G A T A A A G C C A C A T T T G G C T G C C A T G A T G G A T A T T C T C T G +199 T y r P r o A l a L y s P r o T h r L e u T y r T y r L y s A s p L y s A l a T h r P h e G l y C y s H i s A s p G l y T y r S e r L e u

758 +221

759 G A T G G C C C G G A A G A A A T A G A A T G T A C C A A A C T G G G A A A C T G G T C T G C C A T G C C A A G T T G T A A A G C A T C T +222 A s p G l y P r o G l u G l u I l e G l u C y s T h r L y s L e u G l y A s n T r p S e r A l a M e t P r o S e r C y s L y s A 1 a S e r

827 +244

828 T G T A A A G T A C C T G T G A A A A A A G C C A C T G T G G T G T A C A A G G A G A G A G A G T A A A G A T T C A G G A A A A A T T T +245 C y s L y s V a l P r o V a l L y s L y s A l a T h r V a l V a l T y r G l n G l y G l u A r g V a l L y s I l e G l n G l u L y s P h e

896 +267

897 A A G A A T G G A A T G C T A C A T G G T G A T A A A G T T T C T T T C T T C T G C A A A A A T A A G G A A A A G A A G T G T A G C T A T +268 L y s A s n G l y M e t L e u H i s G l y A s p L y s V a l S e r P h e P h e C y s L y s A s n L y s G l u L y s L y s C y s S e r T y r

965 +290

966 A C A G A G G A T G C T C A G T G T A T A G A T G G C A C T A T C G A A G T C C C C A A A T G C T T C A A G G A A C A C A G T T C T C T G +291 T h r G l u A s p A l a G l n C y s I l e A s p G l y T h r I l e G l u V a l P r o L y s C y s P h e L y s G l u H i s S e r S e r L e u

1034 +313

1035 G C T T T T T G G A A A A C T G A T G C A T C C G A T G T A A A G C C A T G C T A A G G T G G T T T T C A G A T T C C A C A T A A A A T G . +314 A l a P h e T r p L y s T h r A s p A l a S e r A s p V a l L y s P r o C y s

1103 +326

1104 T C A C A C T T G T T T C T T G T T C A T C C A A G G A A C C T A A T T G A A A T T T A A A A A T A A A G C T A C T G A A T T T A T T G C 1172 1173 C G C A A A A A A A A A

1184

Fig. 1. The nucleotide sequence and deduced amino acid sequence of human apolipoprotein H (Apo H). The following features are underlined." the five-base nucleotide sequence preceding the initiating methionine codon, which is a four out of five match with the ribosome-binding consensus of Kozak [36], the glycine which is at

position + 1 in the mature protein, the four Asn-X-(Thr/Ser) sites which predict N-linked glycosylation sites, and the AATAAA putative polyadenylation signal. The nucleotide numbering is from the 5' end, the amino acid numbering starts at the amino terminus of the mature protein in accord with Lozier et al. [46]

Amplification of 5' cDNA

Sequence analysis

The c D N A corresponding to the 5' end o f A p o H m R N A was obtained using the RACE procedure [20]. This yielded an additional 69 nucleotides which included 38 nucleotides of 5' untranslated sequence, a four out of five base-pair match with the - 1 to - 5 consensus o f sequences upstream of eukaryotic translational start sites [36], and an initiating methionine.

The combined sequence (Fig. 1) contains an open reading frame of 1035 nucleotides in length followed by a 102-nucleotide 3' untranslated region and 9 bases probably derived from a poly-A tail. It contains a typical AATAAA polyadenylation site [60] at nucleotides 11501155. O f the nucleotides in the open reading frame, 978 encode mature Apo H as determined by comparison with the protein sequence [46].

J.R. Da,, et al.: Molecular cloning and sequence analysis of the cDNA encoding human apolipoprotein H

259

1 MISPVLILFSSFLCHVAIAGRTCPKPDDLPFSTVVPLKTFYEPGEEITYSCKPGYVSRGG

illl II II I

HUMAPOH

II I I

I MISPALIFFSAFLCHVAIAGRR ..............

RATAPOH

61 M R K F I C P L T G L W P I N T L K C T P R V C P F A G I L E N G A V R Y T T F E Y P N T I S F S C N T G F Y L N G A D 23

:I

HUMAPOH

II I IIllllllllill lllilllllill I If I:[III

MWPINTLKCTPRVCPFAGILENGVVRYTTFEYPNTIGFACNPGYYLNGTS

RATAPOH

121 S A K C T E E G K W S P E L P V C A P I I C P P P S I P T F A T L R V Y K P S A G N N S L Y R D T A V F E C L P Q H A M

HUMAPOH

I IIllllllll

II

fill [I II I: II I II I I II II lli: II

73 S S K C T E E G K W S P E L P V C A R I T C P P P P I P K F A A L K E Y K T S V G N S S F Y Q D T V V F K C L P H F A M

RATAPOH

181 FGNDTITCTTHGNWTKLPECREVKCPFPSRPDNGFVNYPAKPTLYYKDKATFGCHDGYSL

HUMAPOH

lllll:lll II

i I lllIiilllillIillllllll

I illll IIII: I I

133 F G N D T V T C T A H G N W T Q L P E C R E V K C P F P S R P D N G F V N Y P A K P V L S Y K D K A V F G C H E T Y K L

RATAPOH

241 DGPEEIECTKLGNWSAMPSCKASCKVPVKKATVVYQGERVKIQEKFKNGMLHGDKVSFFC

HUMAPOH

III11:1111 I

I: i lilIi: illlli:lll llllJ: illll:llill l:i

193 D G P E E V E C T K T G N W S A L P S C K A S C K L S V K K A T V L Y Q G Q R V K I Q D Q F K N G M M H G D K V H F Y C 301 K N K E K K C S Y T E D A Q C I D G T I E V P K C F K E H S S L A F W K T D A S D V K P C

illililliII:

II I l:llli1111111111111111

HUMAPOH

II

253 K N K E K K C S Y T E E A Q C I D G T I E I P K C F K E H S S L A F W K T D A S D V T P C

Fig. 2. An alignment of the amino acid sequences of Apo H from human and rat [3], showing the absence of a 48-residue stretch in the rat sequence that is present near the amino terminus of human Apo H. The amino acids are numbered starting at the initiating

RATAPOH

RATAPOH

methionine in accordance with the numbering of Aoyama et al. [3], for rat Apo H. Identities are indicated by bars (I) and conservative substitutions by colons (:)

Comparison of nucleotide sequence with published amino acid sequence The amino acid sequence deduced from the clone agrees with the amino acid sequence o f the A p o H species determined by Lozier et al. [46], with the exception o f two residues. Residue 102, identified in the sequence determined by Lozier et al. [46] as a cysteine, is predicted to be a serine, and residue 169, originally identified as asparagine and a site o f oligosaccharide attachment, is predicted from the sequence o f our c D N A to be a cysteine. The nucleotide sequence predicts four N-linked glycosylation sites in the A p o H protein, one less than the sequence o f Lozier et al. [46].

9.5-7.5--

4.4-2.4--

1.4--

~

i

Comparison of human and rat nucleotide sequences A c o m p a r i s o n o f the h u m a n A p o H a m i n o acid sequence [46] and the A p o H protein sequence translated f r o m rec o m b i n a n t rat e D N A [3] indicates a 68.7% identity in the c o r r e s p o n d i n g regions encoding m a t u r e A p o H. This identity increases to over 80% when the nucleotide sequences are aligned with the exception o f 144 bases (48 a m i n o acids, Fig. 2) near the 5' end o f the rat A p o H c D N A sequence. Analysis o f the a m i n o acid sequence with the algorithm o f von Heijne [74] and c o m p a r i s o n o f the precursor protein sequence predicted by the c D N A with the a m i n o acid sequence o f the m a t u r e protein, pre-

D~ 1

2

3

4

5

6

7

8

Fig. 3. Northern blot analysis of total RNA from rat liver and various cell lines, hybridized with 32phosphorus-labeled Apo H eDNA insert. Lanes 1-2, 20 lag each of Jurkat cell RNA, THP-1 cell RNA, respectively (negative controls); lanes 3-4, normal rat liver, 5 and 15 lag; lanes 5-6, Hep G2, 5 and 15 lag. Lanes 7-8, Hep 3B, 5 and 15 lag

J.R. Day et al.: Molecularcloning and sequence analysis of the cDNA encoding human apolipoprotein H

260

dicts a signal peptidase cleavage after residue 19 in the amino acid sequence (Fig. 2). A comparison of the 75 sequenced nucleotides in the 3' untranslated portion of the rat Apo H cDNA with the corresponding nucleotides in the human cDNA shows a 69% nucleotide identity in this region.

Blot hybridization analysis and protein secretion analysis Northern blot hybridization analysis of total RNA from Hep G2, Hep 3B, and rat liver revealed one band of approximately 1 kb that hybridizes with the randomprimed Apo H cDNA probe (Fig. 3). This indicates that there is a single class of mRNA present in these cell types. Apo H transcript was not detected in total R N A samples of rat testes, heart, spleen, kidney, lung, stomach, small intestine, and large intestine (data not shown). Jurkat and THP-1 cell lines did not express the Apo H message and therefore served as negative controls for the hybridization conditions being used. Consistent with detection of Apo H message in rat liver was the detection of secreted Apo H in Hep 3B and Hep G2 cell lines using ELISA. Hep 3B and Hep G2 produced Apo H at a rate of 28 ng/h and 26 ng/h per 10 6 cells, respectively.

Discussion Apo H is a structural component of chylomicrons, VLDL, LDL, and HDL [59]. Although its precise metabolic function is not known, Apo H may be involved in lipid metabolism and coagulation since it is associated with lipoproteins, activates lipoprotein lipase [49], and inhibits platelet aggregation [55, 64]. The Apo H locus has been assigned to chromosome 17 (P. Arnaud, M. N. Berkaw, J. R. Day, O. W. McBride, unpublished work). Several apolipoproteins have sequence homologies that parallel their chromosomal loca-

tion in gene clusters (i.e., ApoAI, ApoCIII, and ApoAIV on chromosome 11; ApoE, ApoCI, and Apo CII on chromosome 19) [31]. This suggests that the related apolipoproteins evolved from common genes. Other apolipoproteins are located on independent chromosomes (Apo AII on chromosome 1, Apo B on chromosome 2, and Apo D on chromosome 3) [48]. Since Apo H is found on chromosome 17, and its nucleic acid and protein sequence are not homologous with other apolipoproteins, it appears to belong to the latter group. There are two differences between our deduced amino acid sequence and the protein sequence determined by Lozier et al. [46], both involving cysteines. One difference (position 102, cysteine to serine) eliminates a cysteine and the other change (position 169, asparagine to cysteine) eliminates a potential N-linked glycosylation site and adds a cysteine. These two differences may be due to errors in the protein sequence determination. Another possible explanation for the differences seen between our sequence and the sequence reported by Lozier et al. [46] is that the Apo H protein has been shown to exhibit a genetic polymorphism as demonstrated by analytical isoelectric focusing and immunoblotting [30, 52, 61, 66]. Single-base substitutions, a feature consistent with genetic polymorphisms, have also been described for a number of proteins containing repeating units similar to Apo H, such as complement factor H [15] and GMP-140 [29]. The most striking feature of Apo H is the presence of these repeating sequence units. This structure was described by Lozier et al. [46] as consisting of five complete repeats and part of a sixth repeat. By comparing vertical alignments of amino acids in the repeats from Lozier et al. [46], one position in each repeat unit contained only cysteine, and three other positions contained predominantly cysteine. In addition, there were four cysteines which were not aligned with any other cysteines in the repeats. Additional invariant cysteine positions and fewer unaligned cysteines can be depicted by aligning the

HUMAN GRTCPKPDDLPFSTVVPLK .... TFu .... EPGEEITYS CKPGYVSRGGMRKFI CPLTGLWPI NTLKCT PRVCPFAGILE-NGAVRYT .... TF ..... EYPNTISFSCNTGFYLNGA-DSAKCTEEGKWS PELPVCA PI I CPPPS I PTFATLRVYKPSAGNNS .... LYRDTAVFE CLPQHAMFGN - DT I TCTTHGNWT-KLPECR EVKCPFPSRPD-NGFVNY - PAKPTLu ..... YKDKATFGCHDGu S LDGP-EE IECTKLGNWS-AMPS CK A-S CKVPVKKATVVYQGERVKI QEKFKNGMLHGDKVS FFCKNKEI(~CSYTEDAQCI D-GT- - I EVPKC F RAT GRR MWPINTLKCT PRVCPFAG ILE-NGVVRYT .... TF ..... Eu PNTIGFACNPGYYLNGT-SSSKCTEEGKWS PELPVCA RITCPPPPI PKFAALKEYKTSVGNSS .... FYQDTVVFKCLPHFAMFGN-DTVTCTAHGNWT-Q LPE CR EVKCPFPSRPD-NGFVNu - PAKPVLS ..... YKDKAVFGCHETYKLDGP-EEVECTKTGNWS-ALPS CK A-S CKLSVKKATVLYQGQRVKI QDQFKNGMMHGDKVHFu CKNKEKKCS YTEEAQCI D-GT-- I E I PKC F REPEAT

CONSENSUS 9 9 .CP ................................

Fig. 4. An alignmentof the repeated units present in human and rat Apo H. Five are present in the human sequence, four plus part of a fifth are present in the rat sequence. The repeats are compared to

F.C..GF...G u Y

...... C...G.W

.... P.Co

an overall repeat consensusderived from a large sample of proteins containing this repeat [38]

J.R. Day et al.: Molecular cloning and sequence analysis of the cDNA encoding human apolipoprotein H repeats slightly differently, as in Davie et al. [14]. The alignment of the five repeats with four invariant cysteine positions is presented in Fig. 4. Viewed this way, mature human Apo H consists entirely of these repeating units with the exception of a 19-residue amino acid extension at the carboxyl terminus which terminates in a cysteine. This alignment places one cysteine in the last repeat rather than in one of the the invariant positions. The elements of repeats with invariant cysteines, an additional cysteine in the final repeat, and a 19-residue carboxyl-terminal extension terminating in a cysteine are also present in rat Apo H [3]. The most striking difference between the human and the rat the sequence is a deletion of 48 amino acids in the first repeat unit of the rat protein compared with the human protein. An odd number of cysteine residues are present in the rat molecule because of this deletion. Structures resembling the repeated unit present in human Apo H have been described in over 30 proteins, and approximately 200 examples of this motif have been sequenced. Perkins et al. [58] published a multiple alignment of 101 of the repeats. They are present in diverse classes of proteins, including complement components, regulators of complement activation proteins, a number of membrane-associated receptors and adhesion proteins, and serum proteins (Table 1). With respect to the difference in repeat number between human and rat Apo H, it is of interest to note that there are differences in repeat number between human and mouse C4b-binding protein, human CR1 and the homologous mouse Crry gene product and human and mouse CR2, with the human sequence in each case containing a greater number of repeats. The one segment of Apo H which is not composed of the repeating elements is a 19-residue extension at the carboxyl terminus. Searches of sequence databases with this segment did not reveal any unambiguous and significant similarities. The widespread occurrence of the repeated motif has been noted by a number of authors. Davie et al. [14] noted that a group of 11 protein-binding proteins then known contained these repetitive segments which they called "GP-I repeats" or "GP-I domains". They noted similarities in structure and possibly function between the GP-I repeat, the kringle structures of plasminogen, and the type II structure of fibronectin. Kristensen and Tack [37] grouped a number of proteins, mainly proteins involved in complement activation, into a "superfamily of C3b/C4b-binding proteins". Aegerter-Shaw etal. [1], Kurtz et al. [41], and Paul et al. [56] identified proteins containing the repeat as members of the "complement receptor gene family". The repeat has also been named: "tandem repeating elements" [54], "C3b-C4b regulatory protein repeats" [29], "complement binding protein homologies" [42], "complement receptor repeat elements" [5], "complement regulatory protein repeats" [68], and "short consensus repeat unit" [25]. The need for uniform terminology is quite apparent and fortunately has been recently supplied by Ichinose et al. [27] who called these repeats "sushi structures". A single species o f m R N A of approximately 1 kb was demonstrated by direct hybridization with a random

261

Table 1. Protein containing repeat units similar to apolipoprotein H Protein family

Protein

CompleHuman complement factor B ment/RCA Human complement component proteins CIR Complement component C1S Human complement component C2 Complement component C6 Human complement component C7 Human complement factor I Human complement factor H Mouse complement factor H Human C4b-binding proteins a chains Mouse C4b-binding proteins ~ chains Human C4b-binding protein/~ chain Human complement receptor (CRI) Mouse Crry gene product Human CR2 Mouse CR2 Human decay acceleration factor (DAF) Human membrane cofactor protein (MCP) Other re- Human IL-2 receptor ceptor and Mouse IL-2 receptor adhesion Human granule membrane protein molecules GMP-140 Human endothelial leukocyte adhesion molecule 1 (ELAM-I) Mouse lymphocyte (or lymph node) homing receptor Human lymph node homing receptor (or leukocyte adhesion molecule-1) Serum proteins

Other proteins

subunit/human blood coagulation factor XIII Haptoglobin at chain Rat apolipoprotein H Human apolipoprotein H Cartilage proteoglycan core protein Fibroblast proteoglycan Human thyroid peroxidase Porcine thyroid peroxidase Rat thyroid peroxidase Horseshoe crab coagulation factor C 35-kDa secretory protein of vaccinia

No. of Referrepeats ence 3 2

[53] [44]

2 3 2 2 1 20 20 8

[50] [4] [23] [18] [10] [62] [37] [13]

6

[38]

3 30 5 16 tl 4

[25] [33] [56] [54] [41] [9]

4

[47]

2 2 9

[43] [67] [29]

6

[5]

2

[42] [69] [68] [71]

2

10

[26]

I or 2 [40] 4 [3] 5 [46] 1 1 1 1 1 5 4

[19] [39] [32] [51] [17] [72] [35]

virus

IL, Interleukin; RCA, regulators of complement activation

primed probe from the Apo H c D N A to total R N A from Hep 3B, Hep G2, and rat liver. The probe was also used on total R N A from rat testes, heart, spleen, kidney, lung, stomach, small and large intensine for the purpose of detecting message in these tissues. Only rat liver contained detectable amounts of Apo H m R N A . In summary, we have cloned and sequenced a e D N A for human Apo H (fl-2-Glycoprotein I). H u m a n Apo H was found to be similar to rat Apo H at the e D N A and amino acid level with the exception of a 48-amino acid

262

J.R. Day et al.: Molecular cloning and sequence analysis of the cDNA encoding human apolipoprotein H

deletion at the a m i n o t e r m i n u s in rat. H u m a n A p o H also c o n t a i n s repeating segments similar to those f o u n d in a wide variety of o t h e r proteins.

Acknowledgements. The authors thank Gloria Grimm and Pieter Oort for excellent technical assistance, Dr. S. Baudner (Behringwerke GmbH, Marburg, FRG) for the gift of anti-Apo H antiserum, Dr. Auil Jaiswal, (New York University Medical Center, Department of Cell Biology) for the gift of the human liver eDNA library, and Charlene W Alford (Medical University of South Carolina, Department of Biochemistry) for synthesizing oligonucleotides.

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