©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Site-directed Mutagenesis of Human Lysyl Hydroxylase Expressed in Insect Cells
IDENTIFICATION OF HISTIDINE RESIDUES AND AN ASPARTIC ACID RESIDUE CRITICAL FOR CATALYTIC ACTIVITY (*)

(Received for publication, December 20, 1995; and in revised form, February 5, 1996)

Asta Pirskanen (1) Anne-Maarit Kaimio (1) Raili Myllylä (2) Kari I. Kivirikko (1)(§)

From the  (1)Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, FIN-90220 Oulu and the (2)Biocenter and Department of Biochemistry, University of Oulu, FIN-90570 Oulu, Finland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Lysyl hydroxylase (EC 1.14.11.4), an alpha(2) homodimer, catalyzes the formation of hydroxylysine in collagens. We expressed here human lysyl hydroxylase in insect cells by baculovirus vectors. About 90% of the enzyme produced was soluble 32 h after infection, whereas only 10% was soluble at 72 h. Twelve histidines, five aspartates, and all four asparagines that may act as N-glycosylation sites were converted individually to serine, alanine, or glutamine, respectively, and the mutant enzymes were expressed in insect cells. Three histidine mutations and one aspartate mutation appeared to inactivate the enzyme completely. These and other data suggest that histidines 656 and 708 and aspartate 658 provide the three ligands required for the binding of Fe to a catalytic site, whereas the role of the third critical histidine (residue 706) remains to be established. Three additional histidine mutations also had a major effect, although they did not inactivate the enzyme completely, whereas six further histidine mutations and four out of five aspartate mutations had a much more minor effect. Data on the four asparagine mutations suggested that only two of the potential N-glycosylation sites may be fully glycosylated in insect cells and that one of these carbohydrate units may be needed for full enzyme activity.


INTRODUCTION

Lysyl hydroxylase (EC 1.14.11.4) catalyzes the hydroxylation of lysine in -X-Lys-Gly- sequences in collagens and other proteins with collagen-like sequences. The hydroxylysine residues have two important functions. Their hydroxy groups act as attachment sites for carbohydrate units, and they are essential for the stability of the intermolecular collagen crosslinks (for reviews, see (1) and (2) ). The critical role of hydroxylysine is demonstrated by the marked changes in the mechanical properties of certain tissues seen in patients with the type VI variant of the Ehlers-Danlos syndrome(3, 4, 5) , which is caused by mutations in the gene for lysyl hydroxylase (6, 7, 8, 9, 10) .

Lysyl hydroxylase requires Fe, 2-oxoglutarate, O(2), and ascorbate(1, 2) . The active enzyme is an alpha(2) homodimer with a molecular weight of about 190,000 in gel filtration and a subunit molecular weight of about 85,000 in SDS-PAGE(^1)(11, 12) . Complete cDNA-derived amino acid sequences have now been reported for the enzyme from man (13, 14) and chick embryos(15) .

The catalytic site on lysyl hydroxylase appears to comprise a set of separate locations for binding of the peptide substrate and the various cosubstrates(1, 16) . The Fe is probably bound to the enzyme by three side chains(1, 16) . The 2-oxoglutarate binding site can be divided into distinct subsites: subsite I probably consists of a positively charged side chain of the enzyme which binds the C-5 carboxyl group of the 2-oxoglutarate, and subsite II consists of two cis-positioned coordination sites of the enzyme-bound Fe which is chelated by the C-1-C-2 moiety(1, 16, 17, 18) . The O(2) is thought to be bound end-on in an axial position, and the subsequent decarboxylation of 2-oxoglutarate is assumed to lead to the formation of a ferryl ion, which hydroxylates a lysine residue (1, 16) .

A search for conserved residues within the sequences of several 2-oxoglutarate dioxygenases and a related dioxygenase, isopenicillin N synthase, which also requires Fe, O(2), and ascorbate, indicated a weak homology within two histidine-containing motifs spaced about 50-70 amino acids apart and termed His-1 and His-2 motifs(19) . Recent site-directed mutagenesis studies on the alpha subunit of prolyl 4-hydroxylase have demonstrated that conversion of either of the two conserved histidines present in these motifs to serine inactivates the enzyme completely, suggesting that these residues provide two of the three Fe-binding ligands in prolyl 4-hydroxylase(20) . Furthermore, determination of the crystal structure of isopenicillin N synthase indicated that two of the Fe-binding ligands in this enzyme are the two histidines present in the His-1 and His-2 motifs; the third ligand is an aspartate residue present in position +2 with respect to the histidine in the His-1 motif(21) . A corresponding aspartate is also present in the His-1 motif of lysyl hydroxylase(19) , the two histidines concerned being residues 656 and 708 in the human sequence(13, 19) .

To study the roles of certain histidine and aspartate residues in the catalytic activity of lysyl hydroxylase, we expressed the human enzyme and a number of its mutant forms in insect cells. In addition, the roles of the four glycosylated asparagine residues were studied.


MATERIALS AND METHODS

Construction of Baculovirus Transfer Vectors and Generation of Recombinant Baculoviruses

The transfer vector pVL-LH was constructed by digesting a pBluescript containing a full-length cDNA for human lysyl hydroxylase at the EcoRI site (13) with EagI and EcoRI. The resulting EagI-EcoRI fragment containing 27 base pairs of the 5`-untranslated sequence, the whole coding region, and 724 base pairs of the 3`-untranslated sequence was cloned to the EagI-EcoRI site of the baculovirus transfer vector pVL1392(22) . The recombinant vectors were cotransfected into Spodoptera frugiperda Sf9 cells with a modified Autographa californica virus DNA (Pharmingen) by calcium phosphate transfection, and the recombinant viruses were selected (23) .

Site-directed Mutagenesis

Histidines 88, 225, 241, 474, 517, 536, 613, 656, 657, 700, 706, and 708 (codon CAC or CAT) were converted individually to serine (codon TCC); aspartates 491, 638, 648, 658, and 674 (codon GAC or GAT) to alanine (codon GCA); and asparagines 163, 197, 538, and 686 (codon AAC or AAT) to glutamine (codon CAG). The mutagenesis steps were performed in a pBluescript vector containing the full-length cDNA for human lysyl hydroxylase(13) . The mutagenesis was carried out using an oligonucleotide-directed in vitro mutagenesis system based on the unique site elimination procedure (Pharmacia Biotech Inc.), after which the plasmid was digested with EagI and EcoRI, the resulting fragment was cloned to pVL1392, and the recombinant viruses were generated as above. The sequences were verified by Sanger dideoxynucleotide sequencing (24) .

Analysis of Recombinant Proteins in Insect Cells

The insect cells (Sf9 or High Five, Invitrogen) were cultured as monolayers at 27 °C in TNM-FH medium (Sigma) supplemented with 10% fetal bovine serum (Life Technologies, Inc.). The cells seeded at a density 5 times 10^6 cells/100-mm plate were infected at a multiplicity of 5 with any of the viruses coding for the wild-type lysyl hydroxylase or a mutant type. The cells were harvested 24-72 h after infection, washed with a solution of 0.15 M NaCl and 0.02 M phosphate, pH 7.4, and frozen at -20 °C. The frozen cell pellets were suspended and homogenized in a 1% Nonidet P-40, 0.1 M glycine, and 0.02 M Tris buffer, pH 7.8 (termed Nonidet P-40 buffer), and centrifuged at 10,000 times g for 10 min. The insoluble pellets were further homogenized in a 50% glycerol, 0.6 M NaCl, 1% Nonidet P-40, 0.1 M glycine, 100 µM dithiothreitol, and 0.06 M Tris buffer, pH 7.8 (glycerol buffer), incubated on ice for 30-60 min, and centrifuged at 10,000 times g for 20 min. Aliquots of the supernatants were analyzed by 8% SDS-PAGE under reducing conditions or nondenaturing 8% PAGE and assayed for lysyl hydroxylase activity. The remaining pellets were solubilized in 1% SDS and also analyzed by 8% SDS-PAGE.

Other Assays

Lysyl hydroxylase activity was assayed by a method based on the hydroxylation-coupled decarboxylation of 2-oxo[1-^14C]glutarate(25) . K(m) values were determined as described previously (26) and protein concentrations with a Bio-Rad protein assay kit (Bio-Rad). The levels of expression of the wild-type and mutant enzymes were compared by densitometry of the Coomassie-stained bands in SDS-PAGE using a BioImage instrument (BioImage, Millipore). Western blot analysis was performed using a monoclonal antibody against the purified denatured human lysyl hydroxylase.


RESULTS

Expression of Human Lysyl Hydroxylase in Insect Cells

A baculovirus coding for human lysyl hydroxylase was generated and used to infect High Five cells. The cells were harvested 72 h after infection, homogenized in a buffer containing 1% Nonidet P-40 (Nonidet P-40 buffer, see ``Materials and Methods''), and centrifuged. The cell pellet was homogenized further in a buffer containing 50% glycerol, 0.6 M NaCl, and 1% Nonidet P-40 (glycerol buffer, see ``Materials and Methods''), incubated on ice, and centrifuged. Finally, the remaining pellet was solubilized in 1% SDS, and the samples were analyzed by SDS-PAGE under reducing conditions (Fig. 1).


Figure 1: SDS-PAGE analysis of proteins expressed in High Five cells infected with the recombinant baculovirus for lysyl hydroxylase. Control (lanes 1-3) and lysyl hydroxylase-expressing (lanes 4-6) High Five cells were harvested 72 h after infection and homogenized successively in the Nonidet P-40 and glycerol buffers as described under ``Materials and Methods.'' The samples were run on 8% SDS-PAGE under reducing conditions, and the gels were analyzed by Coomassie staining in panel A and by Western blotting using a monoclonal antibody to human lysyl hydroxylase in panel B. Lanes 1 and 4, 1% Nonidet P-40-soluble proteins; lanes 2 and 5, proteins extracted with the glycerol buffer after the Nonidet P-40 buffer; lanes 3 and 6, proteins solubilized from the remaining pellets with 1% SDS. The arrow indicates the positions of the variably glycosylated lysyl hydroxylase (LH) polypeptides.



The infected cells were found to possess large quantities of a new 85-kDa polypeptide, but little, if any, of this protein could be extracted with the Nonidet P-40 buffer (Fig. 1, lanes 4). Various means of extracting the enzyme were therefore tested. These included sonication, repeated freezing and thawing, and the use of buffers containing high salt concentrations and various detergents (details not shown). Use of a buffer containing 50% glycerol, 0.6 M NaCl, and 1% Nonidet P-40 was found to be the best method of solubilizing the enzyme among all those tested (Fig. 1, lanes 5). This buffer solubilized about 10% of the enzyme produced during the 72 h, the remaining 90% being found in samples solubilized with 1% SDS (Fig. 1, lanes 6). Repeated extractions with the glycerol buffer solubilized only insignificant amounts of additional enzyme. Use of the Nonidet P-40 buffer before the glycerol buffer solubilized large amounts of other proteins, as the protein concentration in the Nonidet P-40 extracts was usually about 0.2 mg/10^6 cells, whereas the concentration in the glycerol extracts was 0.1-0.15 mg/10^6 cells. Densitometric tracing of the Coomassie-stained bands in the glycerol extracts suggested that lysyl hydroxylase represented about 30% of the protein in these extracts, i.e. about 30-50 µg/10^6 cells. Because this soluble enzyme represented about 10% of the total enzyme synthesized, the level of lysyl hydroxylase production appeared to be very high, up to 300-500 µg/10^6 cells. Additional experiments demonstrated that the level of production in the Sf9 cells is only about 10% of that obtained in the High Five cells (details not shown).

To study the time course of the appearance of lysyl hydroxylase in High Five cells, the cells were infected with the virus coding for the enzyme and harvested up to 72 h after infection. After homogenization and extractions, aliquots of the samples were analyzed on SDS-PAGE under reducing conditions (Fig. 2). Lysyl hydroxylase could be detected in SDS-PAGE at 32 h after infection, about 90% of the enzyme being soluble in the glycerol buffer (Fig. 2, A and B, lanes 3). The amount of enzyme in both the glycerol buffer extract and the 1% SDS soluble portion increased up to about 64-72 h, but the increase in the enzyme that was insoluble in the glycerol buffer was much larger than that in the soluble enzyme, so that about half of the total enzyme was soluble at 40 h (Fig. 2, lanes 4), whereas only about 10% was soluble at 72 h (Fig. 2, lanes 7).


Figure 2: Time course of the appearance of recombinant lysyl hydroxylase in High Five cells. The cells were infected with the recombinant lysyl hydroxylase baculovirus and harvested up to 72 h after infection. The cells were homogenized and extracted as described under ``Materials and Methods,'' and the glycerol buffer-soluble (panel A) and 1% SDS-soluble (panel B) proteins were run on 8% SDS-PAGE under reducing conditions and analyzed by Coomassie staining. Lanes 1, control sample (0 h) from noninfected High Five cells; lanes 2-7, protein samples from High Five cells infected with the lysyl hydroxylase-coding virus and harvested 24, 32, 40, 48, 64, and 72 h after infection, respectively. The arrow indicates the positions of the variably glycosylated lysyl hydroxylase (LH) polypeptides.



The catalytic activity was measured with an assay based on hydroxylation-coupled decarboxylation of 2-oxo[1-^14C]glutarate with the synthetic peptide L-1 (Ala-Arg-Gly-Ile-Lys-Gly-Ile-Arg-Gly-Phe-Ser-Gly) as a substrate(25, 27) . A significant level of enzyme activity could already be detected 24 h after infection, the highest activity level being seen at 64 h (Table 1).



K(m) values were determined for Fe, 2-oxoglutarate, ascorbate, and the peptide substrate with the glycerol buffer extract as a source for the enzyme (Table 2). The values were found to be very similar to those reported for lysyl hydroxylase from human placental tissue (28) and chick embryos (26) .



Properties of Histidine to Serine Mutant Lysyl Hydroxylases

Twelve histidines, nine of which are conserved between the human (13) and chick (15) lysyl hydroxylases (underlined in Fig. 3), were converted individually to serine. These included all five histidines present within the highly conserved 76-amino acid region close to the COOH terminus of the polypeptide (residues 639-715), which contains the His-1 and His-2 motifs(13, 19) . The only histidine present in the other highly conserved region (residues 226-286) was likewise subjected to mutation. All of these mutants were expressed in High Five cells. The cells were harvested 48 h after infection and homogenized and extracted as above. The proteins soluble in glycerol buffer were then analyzed by PAGE performed under nondenaturing conditions and by SDS-PAGE, and aliquots of the extracts were used to assay lysyl hydroxylase activity and protein content. None of the mutations was found to inhibit the formation of the enzyme dimer and its polymeric forms (1) when studied by PAGE performed under nondenaturing conditions (details not shown). No differences were found in the mobilities of the various mutant polypeptides in SDS-PAGE, and only minor ones were found in the amounts of the enzyme in the glycerol buffer extracts as judged from the intensities of the Coomassie-stained bands (Fig. 4). These bands were studied by densitometry in each experiment, and the values obtained were used to correct the enzyme activities of the various mutants to the expression level of the wild-type enzyme. In the vast majority of cases this correction was less than ±20%.


Figure 3: Schematic representation of the human lysyl hydroxylase polypeptide. Numbering of the amino acids begins with the first residue in the signal peptide(13) , which is indicated by a shaded box. Only those histidine, aspartate, and asparagine residues that were subjected to mutation in the present experiments are shown. Residues that are conserved between the human (13) and chick (11) lysyl hydroxylase are underlined.




Figure 4: SDS-PAGE analysis of the expression of wild-type or histidine to serine mutant lysyl hydroxylases in High Five cells. The samples extracted with the glycerol buffer were analyzed by 8% SDS-PAGE under reducing conditions and Coomassie staining. Lane 1, extract from noninfected High Five cells; lane 2, extract from cells infected with baculovirus coding for the wild-type lysyl hydroxylase; lanes 3-14, extracts from cells infected with baculoviruses coding for the following histidine to serine mutant lysyl hydroxylases: H88S (lane 3), H225S (lane 4), H241S (lane 5), H474S (lane 6), H517S (lane 7), H536S (lane 8), H613S (lane 9), H656S (lane 10), H657S (lane 11), H700S (lane 12), H706S (lane 13), and H708S (lane 14). The arrow indicates the positions of the variably glycosylated lysyl hydroxylase (LH) polypeptides.



Three histidine mutations, those involving residues 656, 706, and 708, were found to inactivate the enzyme completely, as the activity levels were below that of the noninfected control and below the limit of any accurate assay (Table 3). These three thus included the two histidines (19) suggested by sequence comparisons as possible iron-binding ligands, residues 656 and 708, and a histidine closely flanking residue 708. Mutation of the two additional histidines present in the conserved COOH-terminal region, residues 657 (located at position +1 with respect to histidine 656 and -1 with respect to aspartate 658, see below) and 700, also had a marked effect in that mutation of the former reduced the enzyme activity to about 10% and the latter to 6% (Table 3). Nevertheless, these two mutations did not inactivate the enzyme completely. In addition, mutation of the conserved histidine 517 reduced the enzyme activity to about 10%, whereas mutation of three other conserved histidines, residues 88, 241 (located within a highly conserved region), and 536, had a much more minor effect (Table 3). Mutation of any of the three nonconserved histidines included in this study had either no effect (residue His-225) or reduced the enzyme activity to about 40% (His-474) or 50% (His-613) (Table 3). The K(m) values were determined for Fe, 2-oxoglutarate, and the peptide substrate for all histidine mutant lysyl hydroxylases that were not completely inactive. These values were found to be identical to those with the wild-type enzyme (details not shown).



Properties of Aspartate to Alanine Mutant Lysyl Hydroxylases

Five aspartates, three of which are conserved between the human (13) and chick (15) sequences (underlined in Fig. 3), were converted individually to alanine, and the mutant polypeptides were expressed in High Five cells and analyzed 48 h after infection as described for the histidine mutants. No differences were found in the formation of the enzyme dimer and its polymeric forms or the mobilities of the various mutant polypeptides, and only very minor differences were found in their expression levels (details not shown).

Mutation of aspartate 658, the residue corresponding to the Fe-binding aspartate of isopenicillin N synthase (see the Introduction), appeared to inactivate the lysyl hydroxylase completely (Table 4). Mutation of either of the other two conserved aspartates included in this study reduced the enzyme activity to about 70% (Asp-648) or 40% (Asp-674) of the control value, whereas mutation of the nonconserved Asp-491 consistently increased the enzyme activity to about 170%, and mutation of the other nonconserved Asp-638 (glutamate in the chick sequence) decreased the activity to about 35% (Table 4). As in the cases of the histidine mutations (above), none of the four aspartate mutations (excluding Asp-658) altered the K(m) values of the enzyme (details not shown).



Properties of Asparagine to Glutamine Mutant Lysyl Hydroxylases

The human polypeptide contains four potential N-glycosylation sites(13) , the second through the fourth of these being conserved in the chick enzyme(15) . Some of the carbohydrate units may be needed for maximal enzyme activity, as treatment of lysyl hydroxylase with endoglucosidase H has been found to reduce the catalytic activity to 40-60%(12) . All four of these asparagines were converted individually to glutamines, and the mutant polypeptides were expressed and analyzed as above.

Two asparagine mutations, those involving residues 197 and 538, eliminated the enzyme band in SDS-PAGE with the lowest mobility (Fig. 5, lanes 4 and 5), whereas mutation of asparagines 163 or 686 had no distinct effect on the mobility (Fig. 5, lanes 3 and 6). This suggests that only two of the four potential N-glycosylation sites (Asn-197 and Asn-538) are effectively glycosylated, and thus the multiple bands seen with the wild-type enzyme may mainly correspond to a diglycosylated, monoglycosylated, and nonglycosylated polypeptide. Mutation of the glycosylated Asn-197 decreased the enzyme activity to about 25%, whereas mutation of the other glycosylated Asn-538 or the probably nonglycosylated Asn-163 had no effect on enzyme activity (Table 5). Mutation of the probably nonglycosylated Asn-686 decreased the enzyme activity to about 40% (Table 5), suggesting that a glutamine in this position may have some negative effects, possibly because this residue is larger than asparagine.


Figure 5: SDS-PAGE analysis of the expression of wild-type or asparagine to glutamine mutant lysyl hydroxylases in High Five cells. The samples extracted with the glycerol buffer were analyzed by 8% SDS-PAGE under reducing conditions and Coomassie staining. Lane 1, extract from noninfected High Five cells; lane 2, extract from cells infected with baculovirus coding for the wild-type lysyl hydroxylase; lanes 3-6, extracts from cells infected with baculoviruses coding for the following asparagine to glutamine mutant lysyl hydroxylases: N163Q (lane 3), N197Q (lane 4), N538Q (lane 5), and N686Q (lane 6). The arrow indicates the positions of the variably glycosylated lysyl hydroxylase (LH) polypeptides.






DISCUSSION

The present data indicate that an active human lysyl hydroxylase can be produced by baculovirus vectors and that the K(m) values of the recombinant enzyme for its peptide substrate and cosubstrates are essentially identical to those of the enzyme isolated from vertebrate tissues. Although about 90% of the enzyme produced during the first 32 h of infection could be extracted with the glycerol buffer, about 90% of the enzyme produced during a 72-h infection was in an insoluble form. The level of lysyl hydroxylase production in the High Five cells was exceptionally high compared with those of most proteins in insect cells(29) , and thus one reason for the insolubility may simply be the large quantity of enzyme produced. Lysyl hydroxylase is a luminally oriented peripheral membrane protein in the endoplasmic reticulum(30) , and its complete solubilization from tissues is difficult and requires the use of a high salt buffer containing detergents(25) . These properties of the enzyme may have contributed to its conversion to an insoluble form. In spite of the solubility problems, large amounts of the enzyme could be extracted with the glycerol buffer, and the quantity of enzyme in this extract 48 h after infection appeared to vary only within narrow limits.

The human lysyl hydroxylase polypeptide has 27 histidines, 23 of which are conserved between the human and chick sequences(13) . As it did not seem feasible to mutate all 27 histidines, residues were selected for this purpose in such a way that all five histidines present in the highly conserved COOH-terminal region and the only histidine present in the other conserved region (residues 226-286) were included. The additional residues selected consisted of three further conserved histidines and three nonconserved histidines. This meant that a large percentage of the histidine mutations could be expected to have major effects on catalytic activity.

Three histidine to serine mutations were found to inactivate lysyl hydroxylase completely. These included residues 656 and 708, previously suggested by sequence comparisons as the key residues in the His-1 and His-2 motifs(19) . As the results of recent mutagenesis studies on the alpha subunit of prolyl 4-hydroxylase (20) and crystal structure determination of isopenicillin N synthase (21) have suggested that the corresponding residues in the His-1 and His-2 motifs of these enzymes (19) provide two of the three Fe-binding ligands, it seems very likely that histidines 656 and 708 have a similar function in lysyl hydroxylase. The function of the third critical histidine, residue 706, remains to be established.

Mutation of three additional histidines also had a major effect, although none of these mutations inactivated the enzyme completely. One of these histidines, residue 657, is located between two of the suggested Fe-binding residues, His-656 and Asp-658 (see below), and it is thus not surprising that this mutation had a major effect. His-700 is located in the highly conserved COOH-terminal domain, whereas His-517 is located within a much less well conserved region. Mutation of the three further conserved histidines and the three nonconserved histidines included in this study had much more minor effects.

One of the five aspartate to alanine mutations (residue 658) differed distinctly from the other four in that it inactivated the enzyme completely. This aspartate is located at position +2 with respect to His-656 in the His-1 motif. As an aspartate present in a corresponding position in the His-1 motif of isopenicillin N synthase provides the third Fe-binding ligand in that enzyme, it seems very likely that the three Fe-binding ligands in human lysyl hydroxylase are histidines 656 and 708 and aspartate 658.

Data on the four asparagine to glutamine mutations suggest that only two of the four potential N-glycosylation sites present in the human polypeptide may be effectively used in vivo, at least in insect cells. In agreement with results obtained in the treatment of lysyl hydroxylase with endoglycosidase H(12) , mutation of one of the two effectively glycosylated asparagines reduced the enzyme activity to 25%. The functions of the asparagine-linked carbohydrate units in lysyl hydroxylase are unknown, but present and previous (12) data on lysyl hydroxylase differ from the situation in the closely related enzyme prolyl 4-hydroxylase in that enzymatic removal of the carbohydrate (31) or mutation of the two glycosylated asparagines to glutamines (20) had no effect on the activity of the latter enzyme.


FOOTNOTES

*
This work was supported by grants from the Medical Research Council of the Academy of Finland and FibroGen, Inc., Sunnyvale, CA.

§
To whom correspondence should be addressed: Dept. of Medical Biochemistry, University of Oulu, Kajaanintie 52A, FIN-90220 Oulu, Finland. Tel.: 358-81-537-5801; Fax: 358-81-537-5810.

(^1)
The abbreviation used is: PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Anu Myllymäki and Kirsi Kemppainen for expert technical assistance and Johanna Veijola, M.Sc., for help in producing monoclonal antibodies against lysyl hydroxylase.


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