Cloning of the alpha  Subunit of Prolyl 4-Hydroxylase from Drosophila and Expression and Characterization of the Corresponding Enzyme Tetramer with Some Unique Properties*

Pia Annunen, Peppi Koivunen, and Kari I. KivirikkoDagger

From the Collagen Research Unit, Biocenter and the Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu, Finland

    ABSTRACT
Top
Abstract
Introduction
References

Prolyl 4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens. The vertebrate enzymes are alpha 2beta 2 tetramers, whereas the Caenorhabditis elegans enzyme is an alpha beta dimer, the beta  subunit being identical to protein-disulfide isomerase (PDI). We report here that the processed Drosophila melanogaster alpha  subunit is 516 amino acid residues in length and shows 34 and 35% sequence identities to the two types of human alpha  subunit and 31% identity to the C. elegans alpha  subunit. Its coexpression in insect cells with the Drosophila PDI polypeptide produced an active enzyme tetramer, and small amounts of a hybrid tetramer were also obtained upon coexpression with human PDI. Four of the five recently identified critical residues at the catalytic site were conserved, but a histidine that probably helps the binding of 2-oxoglutarate to the Fe2+ and its decarboxylation was replaced by arginine 490. The enzyme had a higher Km for 2-oxoglutarate, a lower reaction velocity, and a higher percentage of uncoupled decarboxylation than the human enzymes. The mutation R490H reduced the percentage of uncoupled decarboxylation, whereas R490S increased the Km for 2-oxoglutarate, reduced the reaction velocity, and increased the percentage of uncoupled decarboxylation. The recently identified peptide-binding domain showed a relatively low identity to those from other species, and the Km of the Drosophila enzyme for (Pro-Pro-Gly)10 was higher than that of any other animal prolyl 4-hydroxylase studied. A 1.9-kilobase mRNA coding for this alpha  subunit was present in Drosophila larvae.

    INTRODUCTION
Top
Abstract
Introduction
References

Prolyl 4-hydroxylase (proline hydroxylase, EC 1.14.11.2) catalyzes the formation of 4-hydroxyproline in collagens and more than 10 other proteins with collagen-like sequences. This cotranslational and posttranslational modification plays a central role in the synthesis of all collagens because the 4-hydroxyproline residues are essential for formation of the collagen triple helix at body temperature (for reviews, see Refs. 1-3). The vertebrate type I and type II enzymes are (alpha (I))2beta 2 and (alpha (II))2beta 2 tetramers, respectively, in which the beta  subunit is identical to protein-disulfide isomerase (EC 5.3.4.1; PDI)1 and has PDI activity even when present in the tetramer (2-6).

An alpha  subunit of prolyl 4-hydroxylase has also been cloned from the nematode Caenorhabditis elegans (7). This forms an active enzyme in insect cell coexpression experiments with both the C. elegans and human PDI polypeptides, but surprisingly, the enzymes containing the subunit are alpha beta dimers (7, 8). The C. elegans PDI polypeptide also forms an active prolyl 4-hydroxylase with the human alpha  subunits, but these enzymes are alpha 2beta 2 tetramers (8). Assembly of a tetramer or dimer must therefore depend on the properties of the alpha  subunit. It is currently unknown whether other nonvertebrate prolyl 4-hydroxylases are likewise dimers rather than tetramers.

Prolyl 4-hydroxylase requires Fe2+, 2-oxoglutarate, O2, and ascorbate (2, 3). A search for conserved amino acid residues within sequences of several 2-oxoglutarate dioxygenases and prolyl 4-hydroxylase alpha  subunits from various species, together with extensive site-directed mutagenesis studies on the human alpha (I) subunit, have identified five amino acid residues that are critical at the cosubstrate binding sites of prolyl 4-hydroxylases (4, 5, 7, 9-11). Three of these, His-412, Asp-414, and His-483 (numbered according to the human alpha (I) subunit), are involved in binding of the Fe2+ atom, whereas Lys-493 binds the C-5 carboxyl group of 2-oxoglutarate (10, 11). His-501 is an additional critical residue, probably involved in both the binding of the C-1 carboxyl group of 2-oxoglutarate to the Fe2+ atom and the decarboxylation of this cosubstrate (11).

The aim of the present study was to clone the alpha  subunit of prolyl 4-hydroxylase from Drosophila melanogaster and to characterize the corresponding enzyme. Special emphasis was given to comparison of the cDNA-derived amino acid sequence of the Drosophila alpha  subunit with those of the human alpha (I) and alpha (II) subunits and the C. elegans alpha  subunit, to identify conserved and nonconserved residues and regions, and to the question of whether the Drosophila enzyme is an alpha 2beta 2 tetramer or an alpha beta dimer. The amino acid sequence of the Drosophila alpha  subunit was found to show several distinct differences as compared with those of the other species studied, and the Drosophila enzyme was found to have some unique catalytic properties that appear related to these differences.

    MATERIALS AND METHODS

Isolation of cDNA Clones-- A sequence homology search in FlyBase indicated the presence of a 179-nucleotide-long sequence (DM59D7T, FlyBase ID no. FBgn0015713) homologous to those of the vertebrate prolyl 4-hydroxylase alpha  subunits. PCR primers PKA5 (5'-AAATGGAGGTTCCACGGCAG-3') and PKA3 (5'-CCGATCCCATAGTTTGCCAC-CTG-3') were synthesized based on this sequence and used to obtain a 160-bp PCR product from a cDNA pool generated from D. melanogaster mRNA. The purified PCR product was 32P-labeled and used to screen a D. melanogaster larva lambda gt10 cDNA library (CLONTECH). This screening yielded seven positive clones among 600,000 recombinants, two of which, termed Dalpha 2 and Dalpha 6, were characterized in detail.

Nucleotide Sequencing and Sequence Analysis-- The nucleotide sequences were determined by the dideoxynucleotide chain termination method (12) with the Sequenase enzyme (U. S. Biochemical Corp.) in an automated DNA sequencer (Applied Biosystems). Vector-specific or sequence-specific primers (Pharmacia) were used. DNASIS and PROSIS (version 6.0) (Pharmacia) were used to analyze the nucleotide and amino acid sequence data. The cleavage site of the signal pepide was predicted using the computational parameters of von Hejne (13).

Construction of Baculovirus Transfer Vectors and Generation of Recombinant Baculoviruses-- An expression construct for the Drosophila alpha  subunit was obtained by digesting the Dalpha 2 clone with EcoRI. This fragment, which covers the whole coding region, 62 bp of the 5'-untranslated region and 12 bp of the 3'-untranslated region, was ligated to the transfer vector pVL1392 (14).

Arginine 490 (codon CGT) was converted to histidine (CAT) or serine (AGT) to produce the transfer vectors for the recombinant baculoviruses Dalpha H and Dalpha S, respectively. The mutagenesis steps were performed in a pSP72 vector (Promega) containing the Dalpha 2 clone, using a PCR-based method (QuickChange site-directed mutagenesis kit; Stratagene).

To construct a baculovirus transfer vector for the Drosophila PDI polypeptide, a PCR product containing 9 bp of the 5'-untranslated region, the whole coding region, and 227 bp of the 3'-untranslated region, was amplified using sequence-specific (15) primers DRO1 (5'-GTGACCGCAATGAAATTCCTG-3') and DRO4 (5'-GCAAAAAGCTTCGATGGCTAC-3') from a cDNA pool generated from D. melanogaster mRNA, and the fragment was ligated to the SmaI site of the pVL1393.

Spodoptera frugiperda-- Sf9 insect cells (Invitrogen) were cultured as monolayers in TNM-FH medium (Sigma) supplemented with 10% fetal bovine serum (Bioclear) at 27 °C. The recombinant baculovirus transfer vectors were cotransfected into Sf9 cells (Invitrogen) with a modified Autographa californica nuclear polyhedrosis virus DNA (BaculoGold, PharMingen) by calcium phosphate precipitation (16). The resultant viral pools were collected 4 days later, plaque-purified, and amplified (16). The recombinant viruses coding for the Drosophila alpha  subunit, its R490H and R490S mutants, and the Drosophila PDI polypeptide were termed Dalpha , Dalpha H, Dalpha S, and Dbeta . Other recombinant baculoviruses used were human alpha (I), C. elegans alpha , human PDI, and C. elegans PDI coding for the corresponding prolyl 4-hydroxylase subunits (7, 8, 17).

Expression and Analysis of Recombinant Proteins-- Sf9 insect cells were cultured as described above, either as monolayers or in suspension in spinner flasks (Techne Laboratories, Princeton, NJ), and were infected at a multiplicity of 5. For the production of an enzyme tetramer/dimer, the Dalpha , Dalpha H, Dalpha S, human alpha (I), or C. elegans alpha , and the human, C. elegans, or D. melanogaster PDI viruses were used in a ratio 1:1. The cells were harvested 3 days after infection, washed with a solution of 0.15 M NaCl and 0.02 M phosphate, pH 7.4, homogenized in a solution of 0.1 M glycine, 0.1 M NaCl, 10 µM dithiothreitol, 0.1% Triton X-100, and 0.01 M Tris, pH 7.8, and centrifuged at 10,000 × g for 20 min at 4 °C. The resulting supernatants were analyzed by 8% SDS-PAGE or nondenaturing 8% PAGE and assayed for enzyme activity.

Northern Blot Analysis-- Two µg of poly(A)+ RNAs from embryonic, larval, and adult Drosophila (CLONTECH) were run on a 1% formaldehyde gel with molecular weight standards (Promega). The poly(A)+ RNAs were then transferred onto a nitrocellulose filter, and the filter was hybridized under stringent conditions with 32P-labeled Dalpha 2 clone covering the whole coding region of the Drosophila alpha  subunit. Autoradiography time was 9 days.

Other Assays-- Prolyl 4-hydroxylase activity was assayed by a method based on the hydroxylation-coupled decarboxylation of 2-oxo-[1-14C]glutarate as described previously (18), except that in the assays involving the Drosophila enzyme the 2-oxoglutarate concentration was increased from 0.1 to 0.2 µmol/ml and the (Pro-Pro-Gly)10·9H2O peptide substrate concentration from 0.1 to 1.5 mg/ml. The same method was used for measuring the uncoupled decarboxylation activity, except that the peptide substrate was omitted and the amount of enzyme was increased about 10-fold. Km values were determined by varying the concentration of one substrate in the presence of fixed concentrations of the second, whereas the concentrations of the other substrates were held constant (19). The Triton X-100-soluble fraction of the insect cell homogenate was used as the enzyme source for the Km determinations.

The amounts of recombinant proteins were compared by densitometry of Coomassie Brilliant Blue-stained bands in nondenaturing PAGE using a BioImage instrument (BioImage, Millipore).

    RESULTS

Isolation of cDNA Clones-- The first cDNA clone was a 160-bp PCR product obtained from a D. melanogaster cDNA pool with primers PK5 and PK3, which were based on a 179-nucleotide sequence DM59D7T present in FlyBase. This PCR product was used to screen a D. melanogaster larva lambda gt10 cDNA library, and one of the 7 positive clones obtained, Dalpha 2, was found to cover 62 bp of the 5'-untranslated sequence, the whole coding region and 12 bp of the 3'-untranslated sequence of the corresponding Drosophila alpha  subunit mRNA. Dalpha 6 continued 201 bp downward from the 3'-end of Dalpha 2, and thus a total of 213 bp of the 3'-untranslated sequence was characterized. This 3'-untranslated region does not contain the canonical polyadenylation signal AATAAA, but it does contain a GATAAA sequence, which is accompanied 21 bp downstream by a possible poly(A) tail of 4 nucleotides at which the clone ends (these cDNA sequences are not shown but have been deposited in the GenBank/EMBL Data Bank with accession number AF096284).

Amino Acid Sequence of the Drosophila alpha  Subunit and Its Comparison with Those of the Human alpha (I) and alpha (II) Subunits and the C. elegans alpha  Subunit-- The cDNA clone encodes a 535-amino acid polypeptide. A putative signal peptide is present at its N terminus, the most likely first amino acid of the processed alpha  subunit being glutamate, based on the computational parameters of von Hejne (13) and comparison with the N terminus of the processed human alpha (II) subunit (5). Thus the length of the signal peptide is probably 19 amino acids and that of the processed alpha  subunit 516 amino acids (Fig. 1).


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Fig. 1.   Comparison of the amino acid sequence of the alpha  subunit of Drosophila prolyl 4-hydroxylase with those of the human alpha (I), human alpha (II), and C. elegans alpha  subunits. Numbering of amino acids begins with the first residue in the processed polypeptides. Drosophila is indicated by D, human alpha (I) and human alpha (II) by H(I) and H(II), respectively, and C. elegans by C. Gaps (· ·) were introduced for maximal alignment of the polypeptides. Positions of cysteine residues (·) and potential Asn glycosylation sites (---) in any of the polypeptides are indicated above all the sequences. The residues required for the binding of the Fe2+ atom and the C-5 carboxyl group of 2-oxoglutarate as well as the residue that is probably involved in the binding of the C-1 carboxyl group of 2-oxoglutarate and the decarboxylation of this cosubstrate are also indicated (asterisk). White letters on a black background indicate identity, and black letters on a gray background indicate similarity. The following are groups of similar amino acids: G, A, S; A, V; V, I, L, M; I, L, M, F, Y, W; L, K, H; D, E, Q, N; and S, T, E, N.

The Drosophila alpha  subunit is very similar in size to the human alpha (I) and alpha (II) subunits which have 517 (20) and 514 (4) residues, respectively (Fig. 1), whereas the C. elegans alpha  subunit (7) is longer, 542 amino acids, mainly because of a 32-residue C-terminal extension (Fig. 1). The Drosophila alpha  subunit also has a C-terminal extension as compared with the vertebrate alpha (I) and alpha (II) subunits (3), but this extension is much shorter than in the C. elegans alpha  subunit, being only 10 residues (Fig. 1). The Drosophila alpha  subunit sequence has several minor deletions and insertions as compared with the other alpha  subunit sequences, the longest deletion, between residues 225 and 226, being 9 amino acids (Fig. 1).

The overall amino acid sequence identities between the Drosophila alpha  subunit (excluding its 10-residue C-terminal extension) and the human alpha (I) and alpha (II) subunits are 34 and 35%, respectively, whereas its identity to the C. elegans alpha  subunit is 31%. The identity is highest within the catalytically important (2, 3) C-terminal region, residues 385-502 being 51 and 52% identical to those in the human alpha (I) and alpha (II) subunits, respectively, and 46% identical to those in the C. elegans alpha  subunit. All three residues that bind the Fe2+ atom and the lysine that binds the C-5 carboxyl group of the 2-oxoglutarate (11) are conserved, these amino acids being His-402, Asp-404, His-472, and Lys-482 in the Drosophila polypeptide. Surprisingly, however, the fifth critical residue, an additional histidine that is probably involved in both the binding of the C-1 carboxyl group of the 2-oxoglutarate to the Fe2+ atom and the decarboxylation of this cosubstrate (11), is replaced by Arg-490. The recently identified peptide substrate binding domain (21), corresponding to residues 139-229 in the Drosophila alpha  subunit, contains a 9-residue deletion close to its C-terminal end (between residues 225 and 226), whereas residues 139-225 show only 31, 36, and 23% identity to those in the human alpha (I) and alpha (II) subunits and the C. elegans alpha  subunit, respectively.

The 32-amino acid sequence of residues 230-261 shows a particularly low degree of identity to those in the other alpha  subunits, namely 3.1, 6.3, and 12.5% when compared with those of the human alpha (I) and alpha (II) subunits and the C. elegans alpha  subunit (Fig. 1). The sequence of residues 27-60 represents another region of low identity, namely 5.9, 11.8, and 2.9% to the same subunits.

The Drosophila alpha  subunit has three potential attachment sites for asparagine-linked oligosaccharide units, but the positions of these sites are different from those of the two sites (4, 20) present in the human alpha (I) and alpha (II) subunits (Fig. 1). The five cysteine residues present in the vertebrate alpha (I) subunits (2-5, 20) and the C. elegans alpha  subunit (7) are all conserved in the Drosophila alpha  subunit, whereas the vertebrate alpha (II) subunits (4, 5) contain an additional cysteine not present in the other alpha  subunits.

The Drosophila alpha  Subunit Forms Prolyl 4-Hydroxylase alpha 2beta 2 Tetramers with the Drosophila and Human PDI Polypeptides-- A recombinant baculovirus encoding the Drosophila alpha  subunit was generated and used to infect S. frugiperda insect cells. The cells were harvested 72 h after infection, homogenized in a buffer containing Triton X-100, and centrifuged. The cell pellet was solubilized in 1% SDS, and the 0.1% Triton X-100 soluble and 1% SDS soluble proteins were analyzed by 8% SDS-PAGE under reducing conditions followed by Coomassie Blue staining. Most of the insect cell proteins were soluble in the Triton X-100 containing buffer (details not shown). As with prolyl 4-hydroxylase alpha  subunits from other sources, the recombinant polypeptide formed insoluble aggregates, and efficient extraction of the recombinant Drosophila alpha  subunit required the use of 1% SDS. The polypeptide was found in the form of three main bands (Fig. 2, lane 2) with mobilities slightly less than those of the three human alpha (I) subunit bands (Fig. 2, lane 1). Digestion of the sample with endoglycosidase H produced a single band with a mobility slightly higher than that of the lowest of the three bands present in the untreated sample (Fig. 2, lane 3), suggesting that the original polypeptide was present in tri-, di-, and monoglycosylated forms. This agrees with the presence of three potential N-glycosylation sites in the Drosophila alpha  subunit (Fig. 1) and indicates that all three sites are used, at least in insect cells.


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Fig. 2.   SDS-PAGE analysis of expression of the Drosophila prolyl 4-hydroxylase alpha  subunit in insect cells and carbohydrate moieties after endoglycosidase H treatment under reducing conditions. Lanes 1, 2, and 4 show 1% SDS-soluble polypeptides from expressions with baculoviruses coding for the human alpha (I), Drosophila alpha , and C. elegans alpha  subunits, and lane 3 shows the SDS-soluble polypeptides from expression with the Drosophila alpha  subunit baculovirus incubated in the presence of endoglycosidase H. The samples were analyzed by 8% SDS-PAGE and Coomassie Blue staining. Arrows indicate migration of the tri- and monoglycosylated forms of the Drosophila alpha  subunit.

To produce a Drosophila prolyl 4-hydroxylase tetramer or dimer, a cDNA coding for the Drosophila PDI polypeptide was prepared by PCR using primers based on the published sequence (15). A recombinant baculovirus encoding the Drosophila PDI polypeptide was then generated and used to infect insect cells together with the virus coding for the Drosophila alpha  subunit. The cells were harvested 72 h after infection, and Triton X-100-soluble proteins of the cell homogenate were analyzed by nondenaturing 8% PAGE. A Coomassie Blue-stained band with a mobility slightly higher than that of the enzyme tetramer produced by coinfection of insect cells with recombinant viruses coding for the alpha (I) subunit of human prolyl 4-hydroxylase and the human PDI polypeptide (Fig. 3A, lane 1) was seen in samples coinfected with the two types of Drosophila virus (Fig. 3A, lane 2), whereas no such band was seen in a corresponding sample from cells infected with the Drosophila alpha  subunit virus alone (not shown). The mobility of this Drosophila prolyl 4-hydroxylase band is distinctly different from that produced by coinfecting insect cells with baculoviruses coding for the C. elegans alpha  subunit and the human PDI polypeptide (Fig. 3A, lane 5), shown previously (7) to represent an alpha beta dimer. The data thus strongly suggest that the Drosophila prolyl 4-hydroxylase is an alpha 2beta 2 tetramer.


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Fig. 3.   Analysis by PAGE under nondenaturing conditions of prolyl 4-hydroxylase tetramer formation by the Drosophila alpha  subunit with Drosophila or human PDI and by the human alpha (I) subunit with Drosophila PDI when coexpressed in insect cells. Lanes 1-5, Triton X-100-soluble samples are from cells coinfected with viruses coding for human alpha (I) and human PDI (lane 1), Drosophila alpha  and Drosophila PDI (lane 2), Drosophila alpha  and human PDI (lane 3), human alpha (I) and Drosophila PDI (lane 4), and C. elegans alpha  and human PDI (lane 5). The samples were electrophoresed on 8% PAGE under nondenaturing conditions and analyzed by Coomassie Blue staining (A) or by Western blotting using an anti-human PDI polyclonal antibody (B). Arrows marked with T indicate migration of the tetramers; the arrow marked with D, migration of a dimer; and the arrow marked with PDI, migration of free PDI. The prominent band migrating about 75% of the lane distance in A represents a viral protein, and the faint bands below it represent the nonassembled Drosophila (lanes 2 and 4) or human (lanes 1, 3, and 5) PDI polypeptides.

No band corresponding to an enzyme tetramer or dimer could be seen in Coomassie Blue-stained samples from cells coexpressing the Drosophila alpha  subunit and the human PDI polypeptide (Fig. 3A, lane 3), but formation of a small amount of a hybrid enzyme tetramer could be demonstrated by Western blotting with an antibody to the human PDI polypeptide (Fig. 3B, lane 3). Coexpression of the human alpha  subunit with the Drosophila PDI polypeptide produced an enzyme tetramer that could easily be seen by Coomassie Blue staining (Fig. 3A, lane 4).

Catalytic Properties of the Drosophila Prolyl 4-Hydroxylase Tetramer-- Prolyl 4-hydroxylase activity of the Triton X-100-soluble proteins was measured by a method based on the hydroxylation-coupled decarboxylation of 2-oxo-[1-14C]glutarate (18). When the specific activity of the Drosophila enzyme was compared with that of the human type I enzyme, the Coomassie Blue-stained bands in nondenaturing PAGE corresponding to these enzyme tetramers were first studied by densitometry, and the activity levels obtained were corrected (10, 11) for the differences in the amounts of the two types of enzyme. Initial prolyl 4-hydroxylase activity assays performed under standard conditions (18) suggested that the specific activity of the Drosophila enzyme was very low, but when the values were corrected for saturating concentrations of 2-oxoglutarate and the (Pro-Pro-Gly)10 peptide substrate (see below), the specific activity of the Drosophila enzyme increased to about one-third of that of the human type I enzyme (details not shown). The rate of uncoupled 2-oxoglutarate decarboxylation, i.e. decarboxylation observed in the absence of the peptide substrate, was 3.7% of the rate of the complete reaction (details not shown, see also Table II below). This percentage is markedly higher than the value of 0.7% measured for the human type I enzyme (11).

The Km values of the Drosophila enzyme for Fe2+ and ascorbate were very similar to those of the human type I and type II enzymes, whereas the Km for 2-oxoglutarate was about 4-fold (Table I). A major difference was found in the Km for the (Pro-Pro-Gly)10 peptide substrate, because that of the Drosophila enzyme was about 12 times that of the human type I enzyme and 3 times that of the human type II enzyme (Table I). The Drosophila enzyme resembled the vertebrate type II enzymes in being inhibited by poly(L-proline) only at high concentrations, the Ki for poly(L-proline) Mr 7,000 being about 30-fold relative to that obtained for the human type I enzyme and that for poly-(L-proline) Mr 44,000 about 150-fold (Table I). These Ki values of the Drosophila enzyme are not as high as those of the human type II enzyme, however, because the latter values are still about 5-fold and 7-fold higher than the Drosophila enzyme values for poly-(L-proline) Mr 7,000 and 44,000, respectively (Table I).

                              
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Table I
Km values of the Drosophila and human type I and type II prolyl 4-hydroxylases for cosubstrates and the (Pro-Pro-Gly)10 substrate and Ki values for poly-(L-proline)
The values are given as mean ± S.D.

Catalytic Properties of alpha R490H and alpha R490S Mutant Enzyme Tetramers-- Arg-490 in the Drosophila alpha  subunit was converted to either histidine or serine, and baculoviruses coding for the mutant alpha  subunits were used to infect insect cells. The cells were harvested and homogenized as above, and the 1% SDS-soluble proteins were analyzed by 8% SDS-PAGE under reducing conditions followed by Coomassie Blue staining. The expression levels of both types of mutant alpha  subunit per µg of protein were found to be similar to that of the wild-type polypeptide (Fig. 4A).


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Fig. 4.   Analysis by SDS-PAGE under reducing conditions (A) and PAGE under nondenaturing conditions (B) of the expression of Drosophila alpha Arg-490 mutants in insect cells and their tetramer formation. Lanes 1-3 show 1% SDS-soluble proteins from expressions with baculoviruses coding for Drosophila alpha , Drosophila alpha R490H, and Drosophila alpha R490S run on 8% SDS-PAGE under reducing conditions and analyzed by Coomassie Blue staining (A). Arrows indicate migration of the tri-, di-, and monoglycosylated polypeptides. Triton X-100-soluble proteins of the coexpressions of Drosophila alpha  and alpha R490H and of alpha R490S and Drosophila PDI viruses were analyzed on 8% PAGE under nondenaturing conditions with Coomassie Blue staining (B). The arrow marked with T indicates migration of the tetramers.

The mutant alpha  subunits were then coexpressed in insect cells with the Drosophila PDI polypeptide, and the cells were harvested 72 h after infection. Proteins soluble in a Triton X-100 buffer were analyzed by nondenaturing 8% PAGE and assayed for prolyl 4-hydroxylase activity. The Coomassie Blue-stained bands corresponding to the enzyme tetramer (Fig. 4B) were studied by densitometry in each experiment, and these values were used to correct the enzyme activity levels for differences in the amounts of the various types of enzyme tetramer, as described previously (10, 11).

The specific activity of the R490H mutant enzyme tetramer was found to be about 90% that of the wild-type enzyme, whereas the specific activity of the R490S enzyme tetramer was only about 30% (Table II). The rate of uncoupled 2-oxoglutarate decarboxylation as a percentage of the rate of the complete reaction was lower with the R490H mutant enzyme than with the wild-type enzyme, whereas the R490S enzyme gave a very high percentage (Table II). The Km of the R490H mutant enzyme for 2-oxoglutarate was similar to that of the wild-type enzyme, whereas that of the R490S enzyme was about 4-fold that of the wild-type enzyme (Table II).

                              
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Table II
Prolyl 4-hydroxylase activity, rate of uncoupled decarboxylation of 2-oxoglutarate and Km for 2-oxoglutarate of the alpha Arg-490 mutant Drosophila enzymes
The values are given in dpm/50 µl of Triton X-100-soluble cell protein, mean ± S.D. The statistical significances of the values obtained with the mutant enzymes were calculated versus the wild-type enzyme.

Expression of the Drosophila Prolyl 4-Hydroxylase alpha  Subunit mRNA at Different Developmental Stages-- Northern analysis of poly(A)+ RNA from embryonal, larval, and adult Drosophila gave a distinct hybridization signal only with the larval RNA (Fig. 5). The size of the mRNA was about 1.9 kilobases, which agrees well with the size of the 1883 nucleotides characterized by cloning and sequencing.


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Fig. 5.   Northern blot analysis of Drosophila poly(A)+ RNAs. Lanes 1-3 show 2 µg of embryonic, larval, and adult Drosophila poly(A)+ RNA, respectively, hybridized with the 32P-labeled probe covering the coding region of the Drosophila prolyl 4-hydroxylase alpha  subunit. The arrow indicates the 1.9-kilobase band from the larval mRNA. Molecular weight markers are indicated in kilobases.


    DISCUSSION

The data reported here indicate that the alpha  subunit of Drosophila prolyl 4-hydroxylase shows about 30-35% amino acid sequence identity with the two types of human and the C. elegans alpha  subunit. The expression level of the mRNA for the Drosophila alpha  subunit was much higher at the larval stage than the embryonic or adult Drosophila. Our data further indicate that the Drosophila enzyme resembles the vertebrate enzymes (2-4) rather than the C. elegans prolyl 4-hydroxylase alpha beta dimer (7, 8) in that it is an alpha 2beta 2 tetramer.

The Drosophila alpha  subunit was found to have several distinct amino acid sequence differences from those of other species in both its catalytic and peptide substrate binding domains. These differences appear to be related to certain unique catalytic properties of the Drosophila enzyme. The most distinct of these differences was that the histidine likely to assist in the binding of the C-1 carboxyl group of 2-oxoglutarate to the Fe2+ atom, and also in the decarboxylation of this cosubstrate (11), was replaced by Arg-490. Site-directed mutagenesis studies on the human alpha (I) subunit have indicated that replacement of this histidine by other amino acids, including arginine, leads to a 2-3-fold increase in the Km for 2-oxoglutarate, a marked decrease in the reaction velocity, and a marked increase in the rate of uncoupled 2-oxoglutarate decarboxylation as a percentage of the rate of the complete reaction (11). The present data on the wild-type Drosophila enzyme are similar to those obtained with the histidine to arginine mutant human type I enzyme, in that the Km of the former for 2-oxoglutarate was about 4-fold that of the wild-type human enzyme, the reaction velocity was lower, and the percentage of uncoupled decarboxylation was about 5-fold. Mutation of Arg-490 to histidine in the Drosophila alpha  subunit reduced the percentage of uncoupled decarboxylation but did not increase the reaction velocity or reduce the Km for 2-oxoglutarate, whereas mutation of Arg-490 to serine gave a much lower reaction velocity, a much higher percentage of the uncoupled reaction, and an increased Km for 2-oxoglutarate relative to those determined with the wild-type Drosophila enzyme. The data obtained with the R490S mutant Drosophila enzyme agree with those reported for the corresponding human alpha (I) subunit mutant (11), whereas the data obtained with the R490H Drosophila mutant resemble those expected on the basis of the human mutants (11) only in that the rate of uncoupled 2-oxoglutarate decarboxylation was lower than with the wild-type Drosophila enzyme. Thus, other residues in the Drosophila alpha  subunit must influence the environment around residue 490 so that the arginine in this position is much more acceptable than in the human alpha (I) subunit.

The finding that the lysine that binds the C-5 carboxyl group of 2-oxoglutarate (11) is conserved in the Drosophila alpha  subunit is of interest, as the corresponding position in all other 2-oxoglutarate dioxygenases sequenced so far, including lysyl hydroxylase (22-25), is occupied by an arginine (9, 26). It thus seems that this feature is specific to prolyl 4-hydroxylases from various species.

The relatively low degree of identity between the Drosophila alpha  subunit and the other alpha  subunits within the peptide binding domain agrees with the finding that the Km of the Drosophila enzyme for the (Pro-Pro-Gly)10 peptide substrate is higher than that of any vertebrate prolyl 4-hydroxylase tetramer studied (Table I and Ref. 3) or of the C. elegans enzyme dimer (7, 8). It is currently unknown whether this difference is related to possible differences between the sequences of the susceptible prolines in the Drosophila collagens (27-29) and the human collagen substrates. The actual residues in the peptide binding domain that are critical for the binding of (Pro-Pro-Gly)10 and poly-(L-proline) are likewise unknown. The high Km of the Drosophila enzyme for (Pro-Pro-Gly)10 and the high Ki values for poly-(L-proline) indicate that the peptide binding properties of the Drosophila enzyme are more closely related to those of the vertebrate type II enzymes (4, 5) and the C. elegans enzyme (7, 8) than to those of the vertebrate type I enzyme (3).

A 32-amino acid residue region very close to the C-terminal end of the peptide binding domain was found to have a particularly low degree of identity between the Drosophila alpha  subunit and those from other species. No function is currently known for this region, but the low degree of identity suggests that it may represent a variable interdomain sequence. It is also currently unknown whether the catalytic domain begins around residue 262, the C-terminal end of this variable region, or whether there is an additional domain with an unknown function between this region and the catalytic domain, as the highly conserved C-terminal region begins only around residue 385.

The formation of only very small amounts of an enzyme tetramer between the Drosophila alpha  subunit and the human PDI polypeptide is of interest, as the C. elegans alpha  subunit readily forms an active enzyme dimer with the human PDI polypeptide (7) and as the human alpha (I) subunit readily formed an enzyme tetramer with the Drosophila PDI polypeptide. Thus the Drosophila alpha  subunit must have some structural feature that acts against tetramer formation with human PDI, and the Drosophila PDI polypeptide must have, in addition to structural features that allow prolyl 4-hdyroxylase formation with both the human and Drosophila alpha  subunits, some specific feature that is not present in the human PDI polypeptide and that is recognized by the Drosophila alpha  subunit. Further work will be required to elucidate these features, as no specific data are available at present on sequences in these polypeptides that are critical for prolyl 4-hydroxylase assembly.

    ACKNOWLEDGEMENTS

We thank Ari-Pekka Kvist for help with the computational work and Riitta Polojärvi, Jaana Träskelin, Liisa Äijälä, and Eeva Lehtimäki for expert technical assistance.

    FOOTNOTES

* This work was supported by grants from the Research Council for Health within the Academy of Finland.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF096284.

Dagger To whom correspondence should be addressed: Dept. of Medical Biochemistry, University of Oulu, Kajaanintie 52 A, FIN-90220 Oulu, Finland. Tel.: 358-8-537-5801; Fax: 358-8-537-5810.

    ABBREVIATIONS

The abbreviations used are: PDI, protein-disulfide isomerase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; bp, base pair(s).

    REFERENCES
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Abstract
Introduction
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