(Received for publication, December 20, 1995; and in revised form, February 5, 1996)
From the
Lysyl hydroxylase (EC 1.14.11.4), an 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.
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
, and ascorbate(1, 2) . The active enzyme
is an
homodimer with a molecular weight of about
190,000 in gel filtration and a subunit molecular weight of about
85,000 in SDS-PAGE(
)(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
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
, 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
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.
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 cells, whereas the concentration in the glycerol
extracts was 0.1-0.15 mg/10
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
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
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-C]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 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) .
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 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).
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
values of the
enzyme (details not shown).
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.
The present data indicate that an active human lysyl
hydroxylase can be produced by baculovirus vectors and that the K 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 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.