U.S. patent application number 10/068674 was filed with the patent office on 2002-11-28 for alpha2 subunit of prolyl 4-hydroxylase.
Invention is credited to Annunen, Pia Pauliina, Helaakoski, Tarja Inkeri, Kivirikko, Kari I., Nissi, Ritva Kaarina, Nokelainen, Minna Kristiina, Pihlajaniemi, Taina.
Application Number | 20020177203 10/068674 |
Document ID | / |
Family ID | 24541502 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177203 |
Kind Code |
A1 |
Kivirikko, Kari I. ; et
al. |
November 28, 2002 |
Alpha2 subunit of prolyl 4-hydroxylase
Abstract
The present invention relates to novel isoforms of the .alpha.
subunit of prolyl 4-hydroxylase, polynucleotide sequences encoding
these novel proteins, and methods for making such proteins.
Inventors: |
Kivirikko, Kari I.; (Oulu,
FI) ; Pihlajaniemi, Taina; (Oulunsalo, FI) ;
Helaakoski, Tarja Inkeri; (Oulu, FI) ; Annunen, Pia
Pauliina; (Oulu, FI) ; Nissi, Ritva Kaarina;
(Oulu, FI) ; Nokelainen, Minna Kristiina; (Oulu,
FI) |
Correspondence
Address: |
FibroGen, Inc.
Intellectual Property Dept.
225 Gateway Blvd.
South San Francisco
CA
94080
US
|
Family ID: |
24541502 |
Appl. No.: |
10/068674 |
Filed: |
February 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10068674 |
Feb 6, 2002 |
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09686322 |
Oct 10, 2000 |
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09686322 |
Oct 10, 2000 |
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09196581 |
Nov 20, 1998 |
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09196581 |
Nov 20, 1998 |
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08633879 |
Apr 10, 1996 |
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5928922 |
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Current U.S.
Class: |
435/191 |
Current CPC
Class: |
C12N 9/0071
20130101 |
Class at
Publication: |
435/191 |
International
Class: |
C12N 009/02; C12N
009/06 |
Claims
What is claimed is:
1. A polypeptide comprising a human isoform of the a subunit of
prolyl 4-hydroxylase.
2. The polypeptide of claim 1 wherein the a subunit of prolyl
4-hydroxylase is an .alpha.2 subunit.
3. The polypeptide of claim 2 wherein the amino acid sequence of
said polypeptide comprises: (a) the amino acid sequence of SEQ ID
NO:3; (b) fragments of the amino acid sequence of SEQ ID NO:3; or
(c) amino acid derivatives of the amino acid sequence of SEQ ID
NO:3.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 09/686,322, filed Oct. 10, 2000, which is a continuation of
U.S. application Ser. No. 09/196,581, filed Nov. 20, 1998, which is
a divisional of U.S. application Ser. No. 08/633,879, filed Apr.
10, 1996, now U.S. Pat. No. 5,928,922, issued Jul. 27, 1999.
1. INTRODUCTION
[0002] The present invention relates to the identity and
characterization of novel .alpha. subunits of prolyl 4-hydroxylase,
variants thereof; polynucleotide sequences which encode the novel
.alpha.2 subunits of prolyl 4-hydroxylase, and methods for using
and making such novel polynucleotides and polypeptides. The present
invention also relates to the recombinant production of active: (1)
prolyl 4-hydroxylase, or variants thereof, and (2) collagen,
comprising the use of the novel human a subunit of prolyl
4-hydroxylase of the present invention.
[0003] The present invention more specifically relates to
polynucleotides encoding a novel isoform of the a subunit of prolyl
4-hydroxylase, designated the ".alpha.2 subunit," and derivatives
thereof, methods for producing such isoforms or related derivatives
and the use of these proteins and polynucleotides in the production
of recombinant collagen.
2. BACKGROUND
[0004] General Information Regarding Collagen.
[0005] Collagen fibrils, proteoglycan aggregates and glycoproteins
are critical components of the cartilage extracellular matrix that,
collectively, resist compression and the tensile and shear forces
that are generated during articulation. Heineg.ang.rd and Oldberg
(1989) FASEB J. 3:2042-2051; Mayne and Brewton (1993) Cartilage
Degradation: Basic and Clinical Aspects (Woessner, J. F. and
Howell, D. S., eds.) Marcel Dekker, Inc., New York, pp. 81-108.
Mutations in cartilage matrix genes or the genes that encode the
enzymes that affect the biosynthesis, assembly or interactions
between these various matrix components may contribute to
degradation of the cartilage matrix and the loss of normal
cartilage function.
[0006] The Role of Prolyl 4-Hydroxylase in the Production of
Collagen.
[0007] Prolyl 4-hydroxylase plays a crucial role in the synthesis
of all collagens. Specifically, the enzyme catalyzes the formation
of 4-hydroxyproline in collagens and related proteins by the
hydroxylation of proline residues in -Xaa-Pro-Gly-sequences. These
4-hydroxyproline residues are essential for the folding of newly
synthesized collagen polypeptide chains into triple-helical
molecules.
[0008] The vertebrate prolyl 4-hydroxylase is an
.alpha..sub.2.beta..sub.2 tetramer in which the a subunits
contribute to most parts of the catalytic sites. See, Kivirikko et
al. (1989) FASEB J. 3, 1609-1617; Kivirikko et al. (1990) Ann. N.Y.
Acad. Sci. 580, 132-142; Kivirikko et al. (1992) Post Translational
Modifications of Proteins (Harding, J. J. and Crabbe, M. J. C.,
eds.) CRC, Boca Raton, Fla., pp. 1-51. The .beta. subunit has been
cloned from many sources (id.; see also, Noiva and Lennatz (1992)
J. Biol. Chem. 267:6447-49; Freedman et al. (1994) Trends Biochem.
Sci. 19:331-336) and has been found to be a highly unusual
multifunctional polypeptide that is identical to the enzyme protein
disulfide-isomerase (Pihlajaniemi et al. (1987) EMBO J. 6:643-649;
Kojvu et al. (1987) J. Biol. Chem. 262:6447-49), a cellular thyroid
hormone-binding protein (Cheng et al. (1987) J. Biol. Chem.
262:11221-27), the smaller subunit of the microsomal
triacylglycerol transfer protein (Wetterau et al. (1990) J. Biol.
Chem. 265:9800-07), and an endoplasmic reticulum luminal
polypeptide which uniquely binds to various peptides (Freedman,
supra; Noiva et al. (1991) J. Biol. Chem. 266:19645-649; Noiva et
al. (1993) J. Biol. Chem. 268:19210-217).
[0009] A catalytically important .alpha. subunit, designated the
.alpha.1 subunit, has been cloned from human (Helaakoski et al.
(1989) Proc. Natl. Acad. Sci. USA 86:4392-96), chicken (Bassuk et
al. (1989) Proc. Natl. Acad. Sci. USA 86:7382-886) and
Caenorhabditis elegans (Veijola et al. (1994) J. Biol. Chem.
269:26746-753), and its RNA transcripts have been shown to undergo
alternative splicing involving sequences encoded by two
consecutive, homologous 71-bp exons (Helaakoski, supra; Helaakoski
et al. (1994) J. Biol. Chem. 269:27847-854). A second a subunit,
designated the a.sup.2 subunit has been previously obtained from
mouse. Helaakoski et al. (1995) Proc. Natl. Acad. Sci. USA
92:4427-4431.
3. SUMMARY OF THE INVENTION
[0010] The present invention is directed to the cloning and
characterization of human .alpha.-subunit isoforms of prolyl
4-hydroxylase. More specifically, the present invention relates to
human subunit isoforms of the a subunit of prolyl 4-hydroxylase
designated the .alpha.2 subunit, and the polynucleotide sequences
which encode them. Also described herein are methods for producing
the .alpha.2 subunit of prolyl 4-hydroxylase, prolyl 4-hydroxylase
and collagen, wherein said prolyl 4-hydroxylase is comprised of the
.alpha.2 subunit of the present invention and said collagen is
processed into its proper form by such prolyl 4-hydroxylase. In
accordance with the invention, any nucleotide sequence that encodes
the amino acid sequence of claimed .alpha.2 subunit of prolyl
4-hydroxylase can be used to generate recombinant molecules that
direct the expression of human prolyl 4-hydroxylase.
[0011] The present invention is further directed to the use of the
coding sequence for the .alpha.2 subunit of prolyl 4-hydroxylase to
produce an expression vector which may be used to transform
appropriate host cells. The host cells of the present invention are
then induced to express the coding sequence and thereby produce the
.alpha.2 subunit of prolyl 4-hydroxylase, or more generally, in
combination with the p subunit, prolyl 4-hydroxylase.
4. DETAILED DESCRIPTION
[0012] The present invention relates to human .alpha.2 subunits of
prolyl 4-hydroxylase and nucleic acid sequences encoding these
.alpha.2 subunits of the prolyl 4-hydroxylase and derivatives
thereof. In accordance with the invention, any nucleotide sequence
which encodes the amino acid sequence of claimed human .alpha.2
subunit of prolyl 4-hydroxylase can be used to generate recombinant
molecules which direct the expression of prolyl 4-hydroxylase. Also
within the scope of the invention are methods of using and making
these .alpha.2 subunits of prolyl-4hydroxylase.
[0013] a. Definitions
[0014] The term ".alpha.2 subunit of prolyl-4-hydroxylase" refers
to isoforms of the a subunit of prolyl 4-hydroxylase, as encoded by
a single gene as set forth at SEQ ID NO: 3, and genes which contain
conservative substitutions thereto.
[0015] "Active human prolyl 4-hydroxylase" refers to a protein
complex comprising a prolyl 4-hydroxylase .alpha..sub.2.beta..sub.2
tetramer, and may be recombinantly produced.
[0016] The phrase "stringent conditions" as used herein refers to
those hybridizing conditions that (1) employ low ionic strength and
high temperature for washing, for example, 0.015 M NaCl/0.0015 M
sodium citrate/0.1% SDS at 50.degree. C.; (2) employ during
hybridization a denaturing agent such as formamide, for example,
50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%
Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at
pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42.degree. C.; or
(3) employ 50% formamide, 5.times. SSC (0.75 M NaCl, 0.075 M Sodium
citrate), 5.times. Denhardt's solution, sonicated salmon sperm DNA
(50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree. C., with
washes at 42.degree. C. in 0.2.times. SSC and 0.1% SDS.
[0017] The term "purified" as used in reference to prolyl
4-hydroxylase denotes that the indicated molecules are present in
the substantial absence of other biological macromolecules, e.g.,
polynucleotides, proteins, and the like. The term "purified" as
used herein preferably means at least 95% by weight, more
preferably at least 99.8% by weight, of the indicated biological
macromolecules present (but water, buffers, and other small
molecules, especially molecules having a molecular weight of less
than 1000 daltons can be present).
[0018] The term "isolated" as used herein refers to a protein
molecule separated not only from other proteins that are present in
the source of the protein, but also from other proteins, and
preferably refers to a protein found in the presence of (if
anything) only a solvent, buffer, ion, or other component normally
present in a solution of the same. The terms "isolated" and
"purified" do not encompass proteins present in their natural
source.
b. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIGS. 1A and 1B (FIG. 1A, FIG. 1B) set forth the nucleotide
(SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) for the
.alpha.(2) subunit of mouse prolyl 4-hydroxylase.
[0020] FIGS. 2A, 2B, and 2C (FIG. 2A, FIG. 2B, FIG. 2C) set forth
the nucleotide (SEQ ID NO:3) and deduced amino acid sequence (SEQ
ID NO:4) for the .alpha.(2) subunit of human prolyl 4-hydroxylase,
as derived from cDNA clones.
[0021] FIG. 3 (FIG. 3) sets forth the nucleotide (SEQ ID NO:5) and
deduced amino acid sequence (SEQ ID NO:6) for EXON 2 (as identified
in FIG. 2) and flanking intron sequences.
[0022] FIG. 4 (FIG. 4) sets forth the nucleotide (SEQ ID NO:7) and
deduced amino acid sequence (SEQ ID NO:8) for EXON 3 (as identified
in FIG. 2) and flanking intron sequences.
[0023] FIG. 5 (FIG. 5) sets forth the nucleotide (SEQ ID NO:9) and
deduced amino acid sequence (SEQ ID NO:10) for EXON 4 (as
identified in FIG. 2) and flanking intron sequences.
[0024] FIG. 6 (FIG. 6) sets forth the nucleotide (SEQ ID NO:11) and
deduced amino acid sequence (SEQ ID NO:12) for EXON 5 (as
identified in FIG. 2) and flanking intron sequences.
[0025] FIG. 7 (FIG. 7) sets forth the nucleotide (SEQ ID NO:13) and
deduced amino acid sequence (SEQ ID NO: 14) for EXON 6 (as
identified in FIG. 2) and flanking intron sequences.
[0026] FIG. 8 (FIG. 8) sets forth the nucleotide (SEQ ID NO: 15)
and deduced amino acid sequence (SEQ ID NO: 16) for EXON 7 (as
identified in FIG. 2) and flanking intron sequences.
[0027] FIGS. 9A, 9B and 9C (FIG. 9A, FIG. 9B, FIG. 9C) set forth
the nucleotide (SEQ ID NO:17) and deduced amino acid sequence (SEQ
ID NO:18) for EXON 8 (as identified in FIG. 2) and flanking intron
sequences.
c. EXPRESSION Of The .alpha.2 SUBUNIT OF PROLYL 4-HYDROXYLASE OF
THE INVENTION
[0028] (1) Coding Sequences
[0029] In accordance with the invention, polynucleotide sequences
which encode a human isoform of the a subunit of prolyl
4-hydroxylase, or functional equivalents thereof, may be used to
generate recombinant DNA molecules that direct the expression of
the human .alpha.2 subunit of prolyl 4-hydroxylase or its
derivatives, and prolyl 4-hydroxylase comprising the .alpha.2
subunit of prolyl 4-hydroxylase, or a functional equivalent
thereof, in appropriate host cells. Such sequences of an .alpha.2
subunit of prolyl 4-hydroxylase, as well as other polynucleotides
which selectively hybridize to at least a part of such
polynucleotides or their complements, may also be used in nucleic
acid hybridization assays, Southern and Northern blot analyses,
etc.
[0030] Due to the inherent degeneracy of the genetic code, other
nucleic acid sequences which encode substantially the same or a
functionally equivalent amino acid sequence, may be used in the
practice of the invention for the cloning and expression of
.alpha.2 subunit of prolyl 4-hydroxylase proteins. Such nucleic
acid sequences include those which are capable of hybridizing to
the appropriate .alpha.2 subunit of prolyl 4-hydroxylase sequence
under stringent conditions.
[0031] Altered nucleic acid sequences which may be used in
accordance with the invention include deletions, additions or
substitutions of different nucleotide residues resulting in a
sequence that encodes the same or a functionally equivalent gene
product. The nucleic acid product itself may contain deletions,
additions or substitutions of amino acid residues within an
.alpha.2 subunit of the prolyl 4-hydroxylase sequence, which result
in a silent change thus producing a functionally equivalent a
subunit. Such amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues
involved. For example, negatively charged amino acids include
aspartic acid and glutamic acid; positively charged amino acids
include lysine and arginine; amino acids with uncharged polar head
groups having similar hydrophilicity values include the following:
leucine, isoleucine, valine; glycine, alanine; asparagine,
glutamine; serine, threonine; phenylalanine, tyrosine.
[0032] The nucleic acid sequences of the invention may be
engineered in order to alter the .alpha.2 subunit of the prolyl
4-hydroxylase coding sequence for a variety of ends including but
not limited to alterations which modify processing and expression
of the gene product. For example, alternative secretory signals may
be substituted for the native human secretory signal and/or
mutations may be introduced using techniques which are well known
in the art, e.g., site-directed mutagenesis, to insert new
restriction sites, to alter glycosylation patterns,
phosphorylation, etc.
[0033] Additionally, when expressing in non-human cells, the
polynucleotides encoding the prolyl 4-hydroxylase of the invention
may be modified so as to better conform to the codon preference of
the particular host organism.
[0034] In an alternate embodiment of the invention, the coding
sequence of the .alpha.2 subunit of prolyl 4-hydroxylase of the
invention could be synthesized in whole or in part, using chemical
methods well known in the art. See, for example, Caruthers et al.
(1980) Nucleic Acids Symp. Ser. 7:215-233; Crea and Horn (1980)
Nucleic Acids Res. 9(10):2331; Matteucci and Caruthers (1980)
Tetrahedron Letters 21:719; and Chow and Kempe (1981) Nucleic Acids
Res. 9(12):2807-2817. Alternatively, the protein itself could be
produced using chemical methods to synthesize the desired .alpha.2
subunit amino acid sequence at least in part. For example, peptides
can be synthesized by solid phase techniques, cleaved from the
resin, and purified by preparative high performance liquid
chromatography. See, e.g., Creighton (1983) Proteins Structures And
Molecular Principles, W.H. Freeman and Co., New York, pp. 50-60.
The composition of the synthetic peptides may be confirmed by amino
acid analysis or sequencing (e.g., the Edman degradation procedure;
see Creighton (1983) Proteins, Structures and Molecular Principles,
W.H. Freeman and Co., New York, pp. 34-49.
[0035] In order to express the .alpha.2 subunit of prolyl
4-hydroxylase of the invention, the nucleotide sequence encoding
the .alpha.2 subunit of prolyl 4-hydroxylase, or a functional
equivalent, is inserted into an appropriate expression vector,
i.e., a vector which contains the necessary elements for the
transcription and translation of the inserted coding sequence.
[0036] (2) Expression Systems
[0037] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing an .alpha.2
subunit of prolyl 4-hydroxylase coding sequence for prolyl
4-hydroxylase and appropriate transcriptional/translational control
signals. These methods include in vitro recombinant DNA techniques,
synthetic techniques and in vivo recombination. See, for example,
the techniques described in Maniatis et al. (1989) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York and Ausubel et al. (1989) Current Protocols in Molecular
Biology, Greene Publishing Associates and Wiley Interscience, New
York.
[0038] A variety of host-expression vector systems may be utilized
to express a coding sequence of an .alpha.2 subunit of prolyl
4-hydroxylase. These include but are not limited to microorganisms
such as bacteria transformed with recombinant bacteriophage DNA,
plasmid DNA or cosmid DNA expression vectors containing a coding
sequence of an .alpha.2 subunit of prolyl 4-hydroxylase; yeast
transformed with recombinant yeast expression vectors containing a
coding sequence of an .alpha.2 subunit of prolyl 4-hydroxylase;
insect cell systems infected with recombinant virus expression
vectors (e.g., baculovirus) containing sequence encoding the
.alpha.2 subunit of prolyl 4-hydroxylase; plant cell systems
infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing a coding sequence of an .alpha.2 subunit of
prolyl 4-hydroxylase; or animal cell systems infected with
appropriate vectors, preferably semliki forest virus.
[0039] Additionally, the .alpha.2 subunit of prolyl 4-hydroxylase
of the invention may be expressed in transgenic non-human animals
wherein the desired enzyme product may be recovered from the milk
of the transgenic animal. The expression elements of these systems
vary in their strength and specificities. Depending on the
host/vector system utilized, any of a number of suitable
transcription and translation elements, including constitutive and
inducible promoters, may be used in the expression vector. For
example, when cloning in bacterial systems, inducible promoters
such as pL of bacteriophage .lambda., plac, ptrp, ptac (ptrp-lac
hybrid promoter) and the like may be used; when cloning in insect
cell systems, promoters such as the baculovirus polyhedron promoter
may be used; when cloning in plant cell systems, promoters derived
from the genome of plant cells (e.g., heat shock promoters; the
promoter for the small subunit of RUBISCO; the promoter for the
chlorophyll a/b binding protein) or from plant viruses (e.g., the
35S RNA promoter of CaMV; the coat protein promoter of TMV) may be
used; when cloning in mammalian cell systems, promoters derived
from the genome of mammalian cells (e.g., metallothionein promoter)
or from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5 K promoter) may be used; when generating cell
lines that contain multiple copies of an .alpha.2 subunit of prolyl
4-hydroxylase DNA, SV40-, BPV- and EBV-based vectors may be used
with an appropriate selectable marker.
[0040] In bacterial systems a number of expression vectors may be
advantageously selected depending upon the use intended for the
.alpha.2 subunit of the prolyl 4-hydroxylase expressed. For
example, when large quantities of the polypeptides of the invention
are to be produced, vectors which direct the expression of high
levels of protein products that are readily purified may be
desirable. Such vectors include but are not limited to the E. coli
expression vector pUR278 (Ruther et al. (1983) EMBO J. 2:1791), in
which the polypeptide coding sequence may be ligated into the
vector in frame with the lac Z coding region so that a hybrid
AS-lac Z protein is produced; pIN vectors (Inouye and Inouye (1985)
Nucleic Acids Res. 13:3101-3109; Van Heeke and Schuster (1989) J.
Biol. Chem. 264:5503-5509); and the like. pGEX vectors may also be
used to express foreign polypeptides as proteins with glutathione
S-transferase (GST). In general, such proteins are soluble and can
easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. The pGEX vectors are designed to include thrombin
or factor Xa protease cleavage sites so that the cloned polypeptide
of interest can be released from the GST moiety.
[0041] A preferred expression system is a yeast expression system.
In yeast, a number of vectors containing constitutive or inducible
promoters may be used. For a review see Ausubel et al. (1988)
Current Protocols in Molecular Biology, Vol. 2, Greene Publish.
Assoc. & Wiley Interscience, Ch. 13; Grant et al. (1987)
Expression and Secretion Vectors for Yeast, in Methods in
Enzymology, Ed. Wu & Grossman, Acad. Press, New York
153:516-544; Glover (1986) DNA Cloning, Vol. II, IRL Press,
Washington, D.C., Ch. 3; Bitter (1987) Heterologous Gene Expression
in Yeast, in Methods in Enzymology, (Berger and Kimmel, eds.) Acad.
Press, New York 152:673-684; and Strathern et al. (1982) The
Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor
Press, Vols. I and II.
[0042] A particularly preferred system useful for cloning and
expression of the proteins of the invention uses host cells from
the yeast Pichia. Species of non-Saccharomyces yeast such as Pichia
pastoris appear to have special advantages in producing high yields
of recombinant protein in scaled up procedures. Additionally, a
Pichia expression kit is available from Invitrogen Corporation (San
Diego, Calif.).
[0043] There are a number of methanol responsive genes in
methylotrophic yeasts such as Pichia pastoris, the expression of
each being controlled by methanol responsive regulatory regions
(also referred to as promoters). Any of such methanol responsive
promoters are suitable for use in the practice of the present
invention. Examples of specific regulatory regions include the
promoter for the primary alcohol oxidase gene from Pichia pastoris
AOX1, the promoter for the secondary alcohol oxidase gene from P.
pastoris AX02, the promoter for the dihydroxyacetone synthase gene
from P. pastoris (DAS), the promoter for the P40 gene from P.
pastoris, the promoter for the catalase gene from P. pastoris, and
the like.
[0044] Typical expression in Pichia pastoris is obtained by the
promoter from the tightly regulated AOX1 gene. See Ellis et al.
(1985) Mol. Cell. Biol. 5:1111, and U.S. Pat. No. 4,855,231. This
promoter can be induced to produce high levels of recombinant
protein after addition of methanol to the culture. By subsequent
manipulations of the same cells, expression of genes for the
.alpha.2 subunit of prolyl 4-hydroxylase of the invention described
herein is achieved under conditions where a recombinant collagen
protein is adequately hydroxylated by the prolyl 4-hydroxylase of
the present invention and, therefore, can fold into a stable helix
that is required for the normal biological function of the collagen
in forming fibrils.
[0045] Another particularly preferred yeast expression system makes
use of the methylotrophic yeast Hansenula polymorpha. Growth on
methanol results in the induction of key enzymes of the methanol
metabolism, namely MOX (methanol oxidase), DAS (dihydroxyacetone
synthase) and FMHD (formate dehydrogenase). These enzymes can
constitute up to 30-40% of the total cell protein. The genes
encoding MOX, DAS, and FMDH production are controlled by very
strong promoters which are induced by growth on methanol and
repressed by growth on glucose. Any or all three of these promoters
may be used to obtain high level expression of heterologous nucleic
acid sequences in H. polymorpha. The nucleic acid sequence encoding
a .alpha.2 subunit of prolyl 4-hydroxylase of the invention is
cloned into an expression vector under the control of an inducible
H. polymorpha promoter. If secretion of the product is desired, a
polynucleotide encoding a signal sequence for secretion in yeast,
such as the S. cerevisiae prepro-mating factor .alpha.1, is fused
in frame with the coding sequence for the .alpha.2 subunit of the
prolyl 4-hydroxylase of the invention. The expression vector
preferably contains an auxotrophic marker gene, such as URA3 or
LEU2, which may be used to complement the deficiency of an
auxotrophic host.
[0046] The expression vector is then used to transform H.
polymorpha host cells using techniques known to those of skill in
the art. An interesting and useful feature of H. polymorpha
transformation is the spontaneous integration of up to 100 copies
of the expression vector into the genome. In most cases, the
integrated DNA forms multimers exhibiting a head-to-tail
arrangement. The integrated foreign DNA has been shown to be
mitotically stable in several recombinant strains, even under
non-selective conditions. This phenomena of high copy integration
further adds to the high productivity potential of the system.
[0047] In cases where plant expression vectors are used, the
expression of sequences encoding the .alpha.2 subunits of the
invention may be driven by any of a number of promoters. For
example, viral promoters such as the 35S RNA and 19S RNA promoters
of CaMV (Brisson et al. (1984) Nature 310:511-514), or the coat
protein promoter of TMV (Takamatsu et al. (1987) EMBO J. 6:307-311)
may be used; alternatively, plant promoters such as the small
subunit of RUBISCO (Coruzzi et al. (1984) EMBO J. 3:1671-1680;
Broglie et al. (1984) Science 224:838-843); or heat shock
promoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al.
(1986) Mol. Cell. Biol. 6:559-565) may be used. These constructs
can be introduced into plant cells using Ti plasmids, Ri plasmids,
plant virus vectors, direct DNA transformation, microinjection,
electroporation, etc. For reviews of such techniques see, for
example, Weissbach and Weissbach (1988) Methods for Plant Molecular
Biology, Academic Press, New York, Section VIII, pp. 421-463; and
Grierson and Corey (1988) Plant Molecular Biology, 2d Ed., Blackie,
London, Ch. 7-9.
[0048] An alternative expression system which could be used to
express the .alpha.2 subunit of prolyl 4-hydroxylase of the
invention is an insect system. In one such system, Autographa
californica nuclear polyhidrosis virus (AcNPV) is used as a vector
to express foreign genes. The virus grows in Spodoptera frugiperda
cells. Coding sequence for the .alpha.2 subunit of prolyl
4-hydroxylase of the invention may be cloned into non-essential
regions (for example the polyhedron gene) of the virus and placed
under control of an AcNPV promoter (for example, the polyhedron
promoter). Successful insertion of a .alpha.2 subunit of prolyl
4-hydroxylase coding sequence will result in inactivation of the
polyhedron gene and production of non-occluded recombinant virus
(i.e., virus lacking the proteinaceous coat coded for by the
polyhedron gene). These recombinant viruses are then used to infect
Spodoptera frugiperda cells in which the inserted gene is
expressed. (See, e.g., Smith et al. (1983) J. Virol. 46:584; Smith,
U.S. Pat. No. 4,215,051.)
[0049] In mammalian host cells, a number of viral based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, coding sequence for the .alpha.2 subunit prolyl
4-hydroxylase of the invention may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This gene may then be inserted in
the adenovirus genome by in vitro or in vivo recombination.
Insertion in a non-essential region of the viral genome (e.g.,
region E1 or E3) will result in a recombinant virus that is viable
and capable of expressing the polypeptide in infected hosts. (See,
e.g., Logan and Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659.) Alternatively, the vaccinia 7.5 K promoter may be
used. (See, e.g., Mackett et al. (1982) Proc. Natl. Acad. Sci. USA
79:7415-7419; Mackett et al. (1984) J. Virol. 49:857-864; and
Panicali et al. (1982) Proc. Natl. Acad. Sci. 79:4927-4931.)
[0050] Specific initiation signals may also be required for
efficient translation of inserted prolyl 4-hydroxylase coding
sequences. These signals include the ATG initiation codon and
adjacent sequences. In cases where the entire polypeptide gene,
including its own initiation codon and adjacent sequences, is
inserted into the appropriate expression vector, no additional
translational control signals may be needed. However, in cases
where only a portion of a coding sequence is inserted, exogenous
translational control signals, including the ATG initiation codon,
must be provided. Furthermore, the initiation codon must be in
phase with the reading frame of the .alpha.2 subunit of prolyl
4-hydroxylase coding sequence to ensure translation of the entire
insert. These exogenous translational control signals and
initiation codons can be of a variety of origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of appropriate transcription enhancer elements,
transcription terminators, etc. (see Bittner et al. (1987) Methods
in Enzymol. 153:516-544).
[0051] One preferred expression system for the recombinant
production of the .alpha.2 subunit of prolyl 4-hydroxylase of the
invention is in transgenic non-human animals, wherein the desired
polypeptide may be recovered from the milk of the transgenic
animal. Such a system is constructed by operably linking the DNA
sequence encoding the .alpha.2 subunit of the invention to a
promoter and other required or optional regulatory sequences
capable of effecting expression in mammary glands. Likewise,
required or optional post-translational enzymes may be produced
simultaneously in the target cells, employing suitable expression
systems, as disclosed in, inter alia, U.S. application, Ser. No.
8/037,728, operable in the targeted milk protein producing mammary
gland cells.
[0052] For expression in milk, the promoter of choice would
preferably be from one of the abundant milk-specific proteins, such
as alpha S1-casein, or .beta.-lactoglobulin. For example, 5' and 3'
regulatory sequences of alpha S1-casein have been successfully used
for the expression of the human lactoferrin cDNA, and similarly,
the .beta.-lactoglobin promoter has effected the expression of
human antitrypsin gene fragments in sheep milk producing cells.
Wright et al. (1991) Biotechnology 9:830-833. In transgenic goats,
the whey acid promoter has been used for the expression of human
tissue plasminogen activator, resulting in the secretion of human
tissue plasminogen activator in the milk of the transgenics. Ebert
et al. (1991) Biotechnology 9:835-838. Using such expression
systems, animals are obtained which secrete the polypeptides of the
invention into milk. Using procedures well-known by those of the
ordinary skill in the art, the gene encoding the desired prolyl
4-hydroxylase chain can simply be ligated to suitable control
sequences which function in the mammary cells of the chosen animal
species. Expression systems for the genes encoding the .alpha.2
subunit of prolyl 4-hydroxylase are constructed analogously.
[0053] Preferably, the prolyl 4-hydroxylase of the invention is
expressed as a secreted protein. When the engineered cells used for
expression of the proteins are non-human host cells, it is often
advantageous to replace the human secretory signal peptide of the
prolyl 4-hydroxylase protein with an alternative secretory signal
peptide which is more efficiently recognized by the host cell's
secretory targeting machinery. The appropriate secretory signal
sequence is particularly important in obtaining optimal fungal
expression of mammalian genes. For example, in methylotrophic
yeasts, a DNA sequence encoding the in-reading frame S. cerevisiae
.alpha.-mating factor pre-pro sequence may be inserted at the
amino-terminal end of the coding sequence. The .alpha.MF pre-pro
sequence is a leader sequence contained in the .alpha.MF precursor
molecule, and includes the lys-arg encoding sequence which is
necessary for proteolytic processing and secretion (see, e.g.,
Brake et al. (1984) Proc. Natl. Acad. Sci. USA, 81:4642).
[0054] Also preferably, the .alpha.2 subunits of prolyl
4-hydroxylase of the present invention are co-expressed by the host
cell with a .beta. subunit of prolyl 4-hydroxylase and/or collagen,
as described generally in PCT Application No. PCT/US92/09061 (WO
93/07889), such that an .alpha..sub.2.beta..sub.2 prolyl
4-hydroxylase tetramer is formed and this enzyme catalyzes the
formation of 4-hydroxyproline in the expressed collagen.
[0055] Alternatively, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins. Appropriate cells lines or host systems can be chosen
to ensure the correct modification and processing of the foreign
protein expressed. To this end, eukaryotic host cells which possess
the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the gene product
may be used. Such mammalian host cells include but are not limited
to CHO, VERO, BHK, HeLa, COS, MDCK, 293, W138, etc. Additionally,
host cells may be engineered to express various enzymes to ensure
the proper processing of the collagen molecules. For example, the
genes for prolyl 4-hydroxylase (i.e., the gene encoding the a
subunit or prolyl 4-hydroxylase and the gene encoding the .alpha.
subunit of prolyl 4-hydroxylase), may be coexpressed with the
collagen gene in the host cell.
[0056] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express an .alpha.2 subunit of prolyl 4-hydroxylase of
the invention may be engineered. Rather than using expression
vectors which contain viral origins of replication, host cells can
be transformed with .alpha.2 subunit encoding DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of foreign DNA,
engineered cells may be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. The selectable
marker in the recombinant plasmid confers resistance to the
selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express a desired .alpha.2
subunit of prolyl 4-hydroxylase.
[0057] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et al.
(1977) Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase
(Szybalska & Szybalski (1962) Proc. Natl. Acad. Sci. USA
48:2026), and adenine phosphoribosyltransferase (Lowy et al. (1980)
Cell 22:817) genes can be employed in tk.sup.-, hgprt.sup.- or
aprt.sup.- cells, respectively. Also, antimetabolite resistance can
be used as the basis of selection for dhfr, which confers
resistance to methotrexate (Wigler et al. (1980) Natl. Acad. Sci.
USA 77:3567; O'Hare et al. (1981) Proc. Natl. Acad. Sci. USA
78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan and Berg (1981) Proc. Natl. Acad. Sci. USA 78:2072); neo,
which confers resistance to the aminoglycoside G-418
(Colberre-Garapin et al. (1981) J. Mol. Biol. 150:1); and hygro,
which confers resistance to hygromycin (Santerre et al. (1984) Gene
30:147). Recently, additional selectable genes have been described,
namely trpB, which allows cells to utilize indole in place of
tryptophan; hisD, which allows cells to utilize histinol in place
of histidine (Hartman and Mulligan (1988) Proc. Natl. Acad. Sci.
USA 85:8047); and ODC (omithine decarboxylase) which confers
resistance to the omithine decarboxylase inhibitor,
2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L. (1987) In:
Current Communications in Molecular Biology, Cold Spring Harbor
Laboratory).
d. IDENTIFICATION OF TRANSFECTANTS OR TRANSFORMANTS THAT EXPRESS
THE .alpha.2 SUBUNIT PROTEIN OF THE INVENTION AND PURIFICATION OF
THE EXPRESSED PROTEINS
[0058] The host cells which contain the coding sequence and which
express the biologically active gene product may be identified by
at least four general approaches; (a) DNA-DNA or DNA-RNA
hybridization; (b) the presence or absence of "marker" gene
functions; (c) assessing the level of transcription as measured by
the expression of .alpha.2 subunit mRNA transcripts in the host
cell; and (d) detection of the gene product as measured by
immunoassay or by its biological activity.
[0059] In the first approach, the presence of the enzyme coding
sequence inserted in the expression vector can be detected by
DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide
sequences that are homologous to the .alpha.2 subunit of prolyl
4-hydroxylase coding sequence, respectively, or portions or
derivatives thereof.
[0060] In the second approach, the recombinant expression
vector/host system can be identified and selected based upon the
presence or absence of certain "marker" gene functions (e.g.,
thymidine kinase activity, resistance to antibiotics, resistance to
methotrexate, transformation phenotype, occlusion body formation in
baculovirus, etc.). For example, if the .alpha.2 subunit coding
sequence is inserted within a marker gene sequence of the vector,
recombinant cells containing coding sequence of the .alpha.2
subunit of prolyl 4-hydroxylase can be identified by the absence of
the marker gene function. Alternatively, a marker gene can be
placed in tandem with the .alpha.2 subunit sequence under the
control of the same or different promoter used to control the
expression of the .alpha.2 subunit coding sequence. Expression of
the marker in response to induction or selection indicates
expression of the .alpha.2 subunit coding sequence.
[0061] In the third approach, transcriptional activity of the
.alpha.2 subunit coding region can be assessed by hybridization
assays. For example, RNA can be isolated and analyzed by Northern
blot using a probe homologous to the .alpha.2 subunit coding
sequence or particular portions thereof. Alternatively, total
nucleic acids of the host cell may be extracted and assayed for
hybridization to such probes.
[0062] In the fourth approach, the expression of the enzyme product
can be assessed immunologically, for example by Western blots,
immunoassays such as radioimmuno-precipitation, enzyme-linked
immunoassays and the like.
[0063] The expressed enzyme of the invention, which is secreted
into the culture medium, is purified to homogeneity, e.g., by
chromatography. In one embodiment, the recombinant .alpha.2 subunit
of prolyl 4-hydroxylase protein is purified by size exclusion
chromatography.
[0064] However, other purification techniques known in the art can
also be used, including ion exchange chromatography, and
reverse-phase chromatography.
5. EXAMPLES
[0065] The invention will be further understood by reference to the
following examples, which are intended to be purely exemplary of
the invention.
Example 1
Isolation of Mouse CDNA Clones
[0066] A cDNA clone for the mouse .alpha.2 subunit, designated
BT14.1, was obtained from a BALB/c mouse brain cDNA library in
.lambda.gt10 (Clontech, Palo Alto Calif.) by using as a probe, a
cDNA encoding the thymic shared antigen 1, as described in MacNeil,
et al. (1993) J. Immunol. 151:6913-23. The BT14.1 clone had a high
degree of homology to the human and chicken prolyl 4-hydroxylase a
subunit. The cDNA clone BT14.1, however, did not contain sequences
coding for the N-terminal region of the polypeptide. It was
therefore used as a probe to screen mouse brain and skeletal muscle
cDNA libraries.
[0067] Among 600,000 recombinants, 4 positive clones were obtained.
Two of them, M1 and M4 were found to be identical, while M2 had a
deletion and M3 contained two unrelated inserts. The clone M1, was
used to screen 1.6.times.10.sup.6 plaques of a mouse skeletal
muscle cDNA library in .lambda.gt10 (Clontech). One positive clone,
M6, was obtained. This clone was characterized further and was
found to be included in BT14.1. The 5' ends of M1 and BT14.1 were
at the same internal EcoRI site (at nucleotide position 220 of the
sequence shown in FIG. 1). The extreme 5' clone was isolated by
using Ml to screen a mouse skeletal muscle cDNA library, and one
positive clone was obtained, M6. As set forth below, at Example 2,
the cDNA clones, considered in combination, cover the whole coding
region of the mouse .alpha.2 subunit. cDNA clones for the mouse
.alpha.1 subunit were then isolated by screening a 3T3 fibroblast
.lambda.gt11 cDNA library (Clontech) with the human cDNA clone
PA-49 for the .alpha.1 subunit, as described in Helaakoski et al.
(1989) Proc. Natl. Acad. Sci. USA 86:4392-96, and eight positive
clones were obtained out of 600,000 plaques.
[0068] Three of these clones, MA3, MA4, and MA7, were isolated and
sequenced. The nucleotide and predicted amino acid sequences of the
clones showed a significant similarity to those of the human and
chick prolyl 4-hydroxylase a subunit. Two of the clones, MA3 and
MA4, were found to represent the mouse counterparts of human mRNA
containing the alternatively spliced exon 10 sequences, whereas MA7
contained exon 9 sequences. The cDNA clones did not contain the
extreme 5' end of the mRNA. Comparison of the cDNA derived amino
acid sequences with those of the human and chick .alpha.1 subunits
suggests that the cDNA clones cover the whole processed polypeptide
but do not cover the 5' untranslated region or the sequences
corresponding to the N-terminal half of the signal peptide. See,
GenBank database, accession no. U16162.
Example 2
Nucleotide Sequencing, Sequence Analysis, and Northern Blot
Analysis
[0069] The nucleotide sequences for the clones described in Example
1 were determined by the dideoxynucleotide chain-termination
method, as described in Sanger et al. (1977) Proc. Natl. Acad. Sci.
USA 74:5463-67, with T7 DNA polymerase (Pharmacia, Peapack N.J.).
Vector-specific or sequence-specific primers synthesized in an
Applied Biosystems DNA synthesizer (Department of Biochemistry,
University of Oulu) were used. The DNASIS and PROSIS version 6.00
sequence analysis software (Pharmacia), ANTHEPROT (as disclosed in
Deleage et al. (1988) Comput. Appl. Biosci. 4:351-356), the
Wisconsin Genetics Computer Group package version 8 (September
1994), and BOXSHADE (Kay Hofmann, Bioinformatics Group, Institut
Suisse de Recherches Experimentales sur le Cancer Lausanne,
Switzerland) were used to compile the sequence data.
[0070] The cDNA clones cover 2168 not of the corresponding mRNA and
encode a 537-aa polypeptide (FIG. 1). A putative signal peptide is
present at the N terminus of the deduced polypeptide, the most
likely first amino acid of the mature .alpha.2 subunit being
tryptophan, based on the computational parameters of von Hejne
(1986) Nucleic Acid Res. 14:4683-90, which means that the size of
the signal sequence would be 19 aa and that of the processed
.alpha.2 subunit 518 aa. The molecular weight of the processed
polypeptide is 59,000. The cDNA clones also cover 150 bp and 407 bp
of the 5' and 3' untranslated sequences, respectively (FIG. 1). The
3' untranslated sequence contains a canonical polyadenylylation
signal, which is accompanied 12 nucleotides downstream by a poly(A)
tail of 15 nucleotide position.
[0071] The mouse .alpha.2 and mouse .alpha.1 polypeptides are of
similar sizes, .alpha.2 being 518 and .alpha.1 517 amino acids,
assuming that the .alpha.2 polypeptide begins with a tryptophan
residue and .alpha.1 with a histidine residue, as does the human
.alpha.1 polypeptide. The processed human .alpha.1 subunit contains
517 amino acids and the chick .alpha.1 subunit 516 amino acids (as
described in Bassuk, et al., supra), whereas the processed C.
elegans a subunit is longer, 542 aa (Veijola, et al., supra), the
difference being mainly due to a 32 aa extension present in the C
terminus of the polypeptide (FIG. 2).
[0072] The mouse .alpha.2 and .alpha.1 subunits contain two
potential attachment sites for asparagine-linked oligosaccharides;
the positions of the -Asn-Leu-Ser-and -Asn-Glu-Thr- sequences of
the .alpha.2 subunit are indicated in FIG. 1. The positions of the
five cysteine residues present in the human, mouse, and chicken
.alpha.1 subunits and the C. elegans a subunit are all conserved in
the .alpha.2 subunit, but the latter contains an additional
cysteine between the fourth and fifth cysteines of the .alpha.1
subunits. Interestingly, this is located at a site where the
conserved stretch of amino acids is also interrupted in the mouse
.alpha.1 and C. elegans a subunits.
[0073] The overall amino acid sequence identity and similarity
between the mouse .alpha.2 and mouse al subunits are 63% and 83%,
respectively, and those between the mouse .alpha.2 and C. elegans a
subunits are 41% and 67%, respectively, which are almost the same
as between the mouse .alpha.1 and C. elegans a subunits, 43% and
67%. The identity is not distributed equally, however, being
highest within the C-terminal domain, which is believed to
represent the catalytically important part of the .alpha.1 subunit
(id.; Myllyla et al. (1992) Biochem. J. 286:923-927). The two
histidines, residues 412 and 483 in the mouse .alpha.1 subunit
(FIG. 2), that have been suggested to be involved in the Fe.sup.2+
binding sites of prolyl 4-hydroxylase are both conserved and are
both located within the conserved C-terminal domain.
[0074] A mouse multitissue Northern blot (Clontech) containing 2
.mu.g of poly(A)' RNA per sample isolated from various mouse
tissues was hybridized under the stringent conditions suggested in
the manufacture's instructions. The probe used was .sup.32P labeled
cDNA clone BT14.1 or MA7.
[0075] The expression patterns of both types of a .alpha.-subunit
mRNA were found to be very similar, the intensities of the
hybridization signals being highest in the heart, lung, and brain.
The size of the .alpha.2 subunit mRNA was 2.4 kb. The mouse
.alpha.1 subunit was found to have two mRNA transcripts, at least
in the heart, brain, and lungs: the more intense the signal was at
3.4 kb and the weaker one at 4.3 kb.
Example 3
Cell Cultures and Generation of Recombinant Baculoviruses
[0076] Since it was not known initially whether the .alpha.2
polypeptide represented an a subunit of prolyl 4-hydroxylase, a
subunit of prolyl 3-hydroxylase, or some other 2-oxoglutarate
dioxygenase, a recombinant polypeptide was expressed in insect
cells to elucidate its function. Specifically, Spodopiera
frugiperda Sf9 insect cells were cultured at 27.degree. C. in
TNM-FH medium (Sigma-Aldrich, St. Louis Mo.) supplemented with 10%
fetal bovine serum (Invitrogen). To construct an
.alpha.(11)-subunit cDNA for expression, the clone BT14.1 was
digested with the BamHI and EcoRI restriction enzymes, giving a
fragment encompassing bp 592-2168. The 5' fragment was amplified
from the X DNA of M6. The primers used were cDNA specific, M3PH
(5'-AAGTTGCGGCCGCGAGCATCAGC- AAGGTACTGC-3') (SEQ ID NO: 19),
containing an artificial NotI site and M65'PCR
(5'-TCTCCGGATCCAGTTTGTACGTGTC-3') (SEQ ID NO:20), containing a
natural BamHI site. PCR was performed under the conditions
recommended by the supplier of the Taq polymerase (Promega, Madison
Wis.), and the reactions were cycled 27 times as follows:
denaturation at 94.degree. C. for 1 min, annealing at 66.degree. C.
for 1 min, and extension at 72.degree. C. for 3 min. The product
was digested with Not I and BamHI restriction enzymes to give a
fragment that extended from bp 120 to 591. The two Not I-BamHI and
BamNI-EcoRI fragments were then cloned into the PBLUESCRIPT vector
(Stratagene, La Jolla Calif.), the construct was digested with Not
I and EcoRV, and the resulting fragment was ligated into a Not
I-Sma I site of the baculovirus transfer vector pVL1392, wherein
said vector was obtained according to the methods described in
Luckow and Summers (1989) Virology 170:31-39. The pVI construct was
cotransfected into Sf9 insect cells with a modified Autographa
californica nuclear polyhedrosis virus DNA by using the BACULOGOLD
transfection kit (PharMingen, San Diego Calif.). The resultant
viral pool was collected 4 days later, amplified, and plague
purified. The recombinant virus was checked by PCR-based methods,
as described in Malitschek and Schartl (1991) BioTechniques
11:177-178.
Example 4
Expression and Analysis of Recombinant Proteins
[0077] A recombinant baculovirus coding for the mouse .alpha.2
subunit was generated and used to infect S. frugiperda insect cells
with or without the human PDI/.beta. subunit, wherein the insect
cells were infected at a multiplicity of 5. For production of an
enzyme tetramer, the human .alpha.59 1 (see, Vuori, et al., supra)
or mouse .alpha.2 viruses and the PDI/.beta. viruses (id.) were
used in a 1:1 or 2:1 ratio. The cells were harvested 72 hours after
infection, homogenized in 0.01 M tris, pH 7.8/0.1 M NaCl/0.1 M
glycine/10 .mu.M dithiothreitol/0.1% Triton X-100, and centrifuged.
The resulting supematants were analyzed by SDS/8% PAGE or
nondenaturing 7.5% PAGE and assayed for enzyme activities. The cell
pellets were further solubilized in 1% SDS, and the 0.1% Triton
X-100-soluble and 1% SDS-soluble proteins were analyzed by SDS/PAGE
under reducing for the .alpha.1 subunit of prolyl 4-hydroxylase
(Veijola et al., supra; Vuori et al., supra; John et al. (1993)
EMBO J. 2:1587-95). The polypeptide formed insoluble aggregates,
and efficient extraction of the recombinant mouse .alpha.2 subunit
from the cell homogenates required the use of 1% SDS.
Example 5
Enzyme Activity Assays
[0078] Prolyl 4-hydroxylase activity was assayed by a method based
on the decarboxylation of 2-oxoH .sup.14C-glutarate, as disclosed
in Kivirriko and Myllyla (1982) Methods Enzymol. 82:245-304. The
K.sub.m values were determined by varying the concentration of one
substrate in the presence of fixed concentrations of the second
while the concentrations of the other substrates were kept
constant, as set forth in Myllyla et al. (1977) Eur. J. Biochem.
80:349-357.
[0079] The 0.1% Triton X-100 extracts from cell homogenates
containing either the mouse-human type II or the human type I
enzyme were analyzed for prolyl 4-hydroxylase activity with an
assay based on the hydroxylation-coupled decarbosylation of
2-oxo[1.sup.14C]glutarate (Kivirikko and Myllyla, supra). The
activities were very similar for both.
[0080] To show that the activity of the mouse/human type 2 enzyme
was prolyl 4-hydroxylase activity, the amount of 4-hydroxyproline
in a (Pro-Pro-Gly).sub.10 substrate was determined after the
reaction. The values indicated that the type 2 and type 1 enzymes
behaved very similarly and that the activity of the type 2 enzyme
was indeed prolyl 4-hydroxylase activity. The K.sub.m values for
Fe.sup.2+, 2-oxoglutarate, and ascorbate and the K.sub.i value for
pyridine-2,4,-dicarboxylate, which acts as a competitive inhibitor
with respect to 2-oxoglutarate, were likewise highly similar for
the two enzymes, as shown in Table I.
1TABLE I K.sub.m values for cosubstrates and the peptide substrate
and K.sub.1 values for certain inhibitors of the human type 1 and
mouse/human type 2 prolyl 4-hydroxylase tetramers. K.sub.m or
K.sub.i, .mu.M Cosubstrate, substrate, or inhibitor Constant
.alpha.1.sub.2.beta..sub.2 .alpha.2.sub.2.beta..sub.2 Fe.sup.2+
K.sub.m 4 4 2-Oxoglutatrate K.sub.m 22 12 Ascorbate K.sub.m 330 340
(Pro--Pro--Gly) K.sub.m 18 45 Poly(t-proline), M.sub.t 7000 K.sub.i
0.5 300* Poly(t-proline), M.sub.t 44,000 K.sub.i 0.02 30*
Pyridine-2,4-dicarboxylate K.sub.i 2 1 *Values determined as
IC.sub.50.
[0081] Notably, the values differed distinctly in that the type 2
enzyme was inhibited by poly (L-proline) only at very high
concentrations. As poly (L-proline) is a well-recognized, effective
competitive inhibitor of type 1 prolyl 4-hydroxylase from all
vertebrate sources studied and as poly (L-proline) is an effective
polypeptide substrate for all plant prolyl 4-hydroxylases studied.
Such finding was unexpected. Distinct differences thus appear to
exist in the structures of the peptide binding sites of various
prolyl 4-hydroxylases, but no detailed data are currently available
on this aspect.
Example 6
Expression of the Mouse .alpha.2 Subunit and an Active Mouse
.alpha.2 PDI/.beta.Enzyme Tetramer in Insect Cells
[0082] Insect cells were coinfected with two recombinant viruses
coding for the two polypeptides in order to study whether an
association between the mouse .alpha.2 subunit and the human
PDI/.beta.-subunit could be achieved. A hybrid protein was formed
and was soluble in a buffer containing 0.1% Triton X-100, as shown
by PAGE performed under nondenaturing conditions. The mouse
.alpha.2 subunit expressed alone did not give any extractable
recombinant protein under the same conditions, termed here the type
1 tetramer, indicating that the hybrid protein is likely to be an
.alpha.2.sub.2.beta..sub.2 tetramer, termed the type 2 tetramer. No
difference was found in the association of the .alpha.2 and
.alpha.1 subunits with the PDI/.beta. subunit into the tetramer. To
show that the hybrid protein formed contains the human PDI/.beta.
subunit, Western blotting was performed. When the mouse .alpha.2
subunit was expressed together with the human PDI/.beta. subunit,
the protein complex contained the PDI/.beta. subunit.
Example 7
Isolation and Sequencing of Human .alpha.2 Subunit Gene
[0083] A human lung fibroblast genomic library (cloned in the lamda
FIX vector (Stratagene)) and a human chromosome 5 library (cloned
in the lamda vector Charon 40 (American Type Culture Collection,
Manassas Va.)) were screened with probes comprising
.sup.32P-labelled nick-translated PCR fragments corresponding to
the previously characterized human prolyl 4-hydroxylase a subunit
cDNA sequence.
[0084] Positive clones from both the human lung fibroblast library
and the human chromosome 5 library were identified, isolated and
analyzed by southern blotting. Suitable fragments were subcloned
into pSP72 vector (Promega) for further analysis.
[0085] Five positive clones, designated GL-2, GL-5, GL-20, GL-141
and GL-142 were obtained from the human lung fibroblast genomic
library. Two of these clones, GL-2 and GL-141 were identical.
Clones corresponding to the 5' and 3'-ends of the gene encoding the
.alpha.2 subunit of prolyl 4-hydroxylase were not obtained.
[0086] The human chromosome 5 library was screened twice with two
separate probes. The first probe corresponded to the 5'-end of the
previously characterized cDNA sequence for .alpha.2 subunit of
prolyl 4-hydroxylase. The second probe corresponded to the 3'-end
of the same cDNA sequence. Several positive clones were obtained,
including GL-3, GL-4, GL-9, GL-11, GL-11B, and GL-156GL-3, GL-4,
GL-9 and GL-11B corresponded to the 5'-end of the protein. GL-11A
and GL-156 corresponded to the 3'-end of the protein clones GL-11A
and GL-156 were found to be identical.
[0087] The derived sequence corresponding to the gene is more than
30 kb in size and is comprised of 15 exons. The exons that encode
solely protein sequences vary from 54 to 240 base pairs and the
introns vary from 241 to at least 3200 base pairs (see, FIGS.
2-9).
[0088] As compared to the gene sequence for the .alpha.1 subunit,
only one exon of the .alpha.2 subunit corresponds to the two
mutually exclusive spliced exons of the a l subunit gene (EXON 9 of
the .alpha.1 subunit gene).
[0089] The deduced amino acid sequence is 63% homologous to the
known .alpha.(1) subunit.
Example 8
Expression of the Human .alpha.2 Subunit of Prolyl 4-Hydroxylase in
Insect Cells
[0090] Using the methods of Examples 3, 4 and 6, the .alpha.2
subunit isoform of prolyl 4-hydroxylase was expressed and analyzed.
Expression data in insect cells demonstrated that the .alpha.2
subunit isoform forms an active type 2 prolyl-4-hydroxyl
.alpha..sub.2.beta..sub.2 tetramer with the human .beta.
subunit.
[0091] Various modifications of the invention, in addition to those
shown and described herein, will become apparent to those skilled
in the art from the foregoing description. Such modifications are
intended to fall within the scope of the appended claims. It is
also to be understood that all base pair sizes given for
nucleotides are approximate and are used for purposes of
description.
[0092] All references cited herein are hereby incorporated by
reference in their entirety.
Sequence CWU 1
1
* * * * *