(Received for publication, July 15, 1994; and in revised form, June 26, 1995)
From the
The properties of a mouse liver copper binding protein (CuBP)
and human placental S-adenosylhomocysteine hydrolase (SAHH)
were compared to test the hypothesis that CuBP is SAHH. CuBP and SAHH
migrated identically on SDS-polyacrylamide gel electrophoresis gels,
and their 48-kDa monomers both self-associate to tetramers. Human
placental SAHH cross-reacted with polyclonal antibodies to mouse liver
CuBP, and CuBP from mouse liver cross-reacted with two monoclonal
antibodies to human placental SAHH. A third monoclonal antibody to
human placenta SAHH reacted weakly with the mouse liver protein but
well with CuBP from human lymphoblasts. NAD-activated
CuBP has high SAHH enzymatic activity. Moreover, human placental SAHH,
like mouse liver CuBP, has a single high affinity copper binding site
per 48-kDa subunit. Thus, the data confirm that CuBP is SAHH, and SAHH
is proposed to be a bifunctional protein with roles in sulfur-amino
acid metabolism and copper metabolism. The copper binding activity of
SAHH is proposed to play a significant role in the intracellular
distribution of copper, and SAHH enzymatic activity may influence
copper metabolism through its role in cysteine biosynthesis from
methionine.
A copper binding protein (CuBP) ()was recently
purified from mouse liver(1) . The 48-kDa subunit of CuBP
self-associates to a dimer and tetramer(1) . CuBP has a single,
high affinity copper-binding site per 48-kDa monomer(2) . CuBP
was proposed to be a major copper binding protein in mouse liver
cytosols because it coeluted with high copper binding fractions from
HPLC columns and had a higher affinity for copper than other proteins
in those fractions(1) . A possible role for CuBP in
intracellular copper metabolism was inferred from the abnormal
properties of CuBP that were isolated from the livers of brindled
mice(3) . The brindled mouse is an animal model (4) for Menkes disease that is a fatal X-linked disease of
copper metabolism(5, 6, 7) . Candidate genes
(cDNAs) for the defects in Menkes disease and Wilson's disease
were recently
cloned(8, 9, 10, 11, 12, 13, 14) ,
which are homologous to bacterial membrane copper and cadmium
transporters(15, 16) . Based on these homologies and
the characteristics of these diseases, the proteins encoded by these
genes may be intracellular membrane copper transport
proteins(8) . Our working hypothesis is that CuBP interacts
with these proteins in copper metabolism.
The cDNA for CuBP was recently cloned from a mouse liver cDNA library(17) . The deduced amino acid sequence of CuBP (17) showed greater than 95% identities to the deduced amino acid sequences of rat (18) and human (19, 20) S-adenosylhomocysteine hydrolase (SAHH). Human placental and rat liver SAHH are 48-kDa proteins that self-associate to tetramers(21, 22) . SAHH catalyzes the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and homocysteine(23) . SAHH activity may be required in copper metabolism for the synthesis of cysteine from homocysteine. Cysteine is required in large amounts for the synthesis of metallothionein (24) and glutathione, which, in turn, have postulated roles in copper metabolism.
The cDNA encoding CuBP was cloned by polymerase chain reaction and library hybridization screening based on the partial sequence of a cyanogen bromide peptide from CuBP(17) . While the sequence of the cDNA that was cloned clearly indicated that it was the cDNA encoding SAHH, that alone did not prove that CuBP was SAHH. The molecular and functional properties of the mouse liver CuBP and human placental SAHH are compared here to further test the hypothesis that CuBP is SAHH. Since the results are consistent with this hypothesis, SAHH is proposed to be a bifunctional protein with roles in methionine/cysteine metabolism and copper metabolism.
Figure 1:
Silver-stained SDS-PAGE analysis of
mouse liver CuBP/SAHH and human placental and recombinant CuBP/SAHH. Lanes1 and 8 are molecular weight markers;
-galactosidase, 116 kDa; phosphorylase b, 97.4 kDa;
bovine serum albumin, 66 kDa; ovalbumin, 45 kDa;
glyceraldehyde-3-phosphate dehydrogenase, 36 kDa; carbonic anhydrase,
29 kDa; and trypsinogen, 24 kDa. Lanes2 and 7, 300 ng of mouse liver CuBP/SAHH (labeled M); lanes3 and 4, 300 ng of human recombinant
placental CuBP/SAHH (labeled R); and lanes5 and 6, 250 ng of human placental CuBP/SAHH (labeled P).
Figure 2:
Autoradiograph of Western blot of mouse
liver and human placental and recombinant CuBP/SAHH using polyclonal
antibodies raised to mouse liver CuBP/SAHH. The Western blot was
incubated with a 1:300 dilution of rabbit antisera to mouse liver CuBP.
Bound antibodies were detected with I-protein A. Lane1, 300 ng of mouse liver CuBP/SAHH (labeled M); lane2, 300 ng of human recombinant placental
CuBP/SAHH(labeled R); and lane3, 250 ng of
human placental CuBP/SAHH (labeled P).
Figure 3: Western blot analysis, using monoclonal antibodies raised to human placental CuBP/SAHH, of mouse liver CuBP/SAHH, human lymphoblast CuBP/SAHH, and human placental and recombinant CuBP/SAHH. Bound antibody was detected with antimouse antibody alkaline phosphatase conjugate (diluted 1:8,000) and the substrates nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate as described under ``Experimental Procedures.'' A, the immunoblot was incubated with a 1:10,000 dilution of monoclonal antibody 1. Lane1, 220 ng of mouse liver CuBP/SAHH (labeled M); lanes2 and 3, 220 ng of human recombinant CuBP/SAHH (labeled R); and lanes4 and 5, 190 ng of human placental CuBP/SAHH (labeled P). B, the immunoblot was incubated with a 1:10,000 dilution of monoclonal antibody 2. Lane1, 220 ng of mouse liver CuBP/SAHH (labeled M); lane2, 220 ng of brindled mouse liver CuBP/SAHH (labeled Br); lane3, 220 ng of human recombinant CuBP/SAHH (labeled R); and lane4, 190 ng of human placental CuBP/SAHH (labeled P). C, the immunoblot was incubated with a 1:10,000 dilution of monoclonal antibody 3. Lane1, 220 ng of mouse liver CuBP/SAHH (labeled M); lane2, 220 ng of brindled mouse liver CuBP/SAHH (labeled Br); lane3, 220 ng of human recombinant CuBP/SAHH (labeled R); and lane4, 190 ng of human placental CuBP/SAHH (labeled P). D, the immunoblot was incubated with a 1:10,000 dilution of monoclonal antibody 3. Lanes1 and 2, partially purified and concentrated human lymphoblast CuBP/SAHH (labeled L); lane3, 220 ng of human recombinant CuBP/SAHH (labeled R).
Figure 4:
SAHH activity of mouse liver CuBP/SAHH.
SAHH activity was measured in the synthesis direction in 50 mM potassium phosphate, pH 7.0, 1 mM DTT. 1.36 µg of
mouse liver CuBP/SAHH was incubated at 37 °C with 5 mM homocysteine and 50 µM [8-C]adenosine for the times indicated. The opencircles represent the reaction without any
additions. The closedcircles represent the reaction
with five equivalents of NAD
. The opensquares represent the reaction with five equivalents
NAD
and two equivalents
CuSO
.
Since SAHH has a high affinity for copper(2) , the effect of copper on the catalytic activity of SAHH was examined in vitro. The addition of 2 molar equivalents of copper had no significant effect on the catalytic activities of either mouse liver CuBP (Fig. 4) or human placental SAHH (data not shown) under the assay conditions used.
Figure 5:
Cu binding to human
recombinant SAHH in the presence of EDTA. SAHH (0.3-2.1
µM) was incubated with 0.2 µM EDTA and 0.25
µM
Cu.
Cu-SAHH was separated
from
Cu-EDTA and
Cu by ultrafiltration. The
amount of
Cu-SAHH was measured and subtracted from the
total SAHH added to calculate the free SAHH. The opencircles represent the means ± S.D. of triplicates
of the corrected observed data. The closedcircles represent the calculated amount bound from the best fit of the
data to the binding equation. The best fit K
was 5.8 ± 0.4
10
M.
The deduced >95% amino acid sequence identities of mouse liver CuBP, rat liver SAHH, and human placental SAHH (17) clearly indicated that the cDNA cloned was the cDNA for mouse liver SAHH. However, the sequence identity alone did not unequivocally establish that CuBP is SAHH. The possibilities were that 1) CuBP and SAHH were copurified, and the amino acid sequence of the cyanogen bromide peptide that was used for cloning the cDNA was from SAHH rather than CuBP; 2) CuBP and SAHH shared at least one homologous domain and also contained non-homologous domains; or 3) that mouse CuBP was indeed a homologue of rat and human SAHH.
SAHH as a contaminant
of CuBP purification seemed unlikely because the cyanogen bromide
peptide used was not a minor peptide. Moreover, CuBP purification
required three different types of purification
steps(1, 2) , i.e. Mono Q, chelating
Superose, and phenyl Superose chromatography. Though possible, it seems
improbable that both SAHH and CuBP would have similar isoelectric
points, similar amounts and distributions of surface histidines, and
similar surface hydrophobicities that these HPLC steps require and also
have the same monomeric molecular weights and yet be two different
proteins. Also, the K for copper binding
to mouse liver CuBP/SAHH is similar to the K
reported here for human SAHH (2) . If copper binding
by CuBP were only due to contaminating SAHH, their measured K
values would be very different,
especially because the protein concentration was varied rather than
copper concentration in the binding method used.
Because CuBP and SAHH comigrated on SDS-PAGE and both oligomerized to a tetramer of the same molecular weight, it seemed highly unlikely that they only shared a homologous domain. Also, if CuBP and SAHH shared only one homologous domain, then at least two cDNAs in the cDNA library should have hybridized to SAHH-specific probes, but only one was detected(17) . However, it was possible that the CuBP cDNA was in much lower abundance than the SAHH cDNA in the library used for cloning. Therefore, it was possible that although the cDNA cloned was clearly the cDNA encoding SAHH, it was not the gene for mouse liver CuBP.
Since none of the above possibilities could be unequivocally excluded, it was imperative to compare the physical and functional properties of the purified protein that was first identified as a CuBP with the protein identified as SAHH. Polyclonal antibodies raised to mouse liver CuBP cross-reacted with human placental and recombinant SAHH. Since polyclonal antibodies react with numerous epitopes, cross-reactivity to proteins that share a domain is expected. Also, if SAHH had copurified with CuBP, the antibodies would react with SAHH. Therefore, cross-reactivity with polyclonal antibodies did not unambiguously indicate that CuBP was SAHH. However, cross-reactivities of similar magnitudes with three different monoclonal antibodies is less likely with two similar but non-identical proteins. Because it is not known which epitopes on SAHH the monoclonals react with, it is possible, though unlikely, that all three monoclonals reacted with the same epitopes on a putative homologous domain. However, one of the monoclonal antibodies recognized one of the few epitopes that was unique to the human protein, indicating that this monoclonal antibody recognized a different epitope than the other two monoclonal antibodies.
Most important, CuBP and SAHH share the same functional
properties, i.e. both proteins have a high affinity for copper
and catalyze the synthesis of S-adenosylhomocysteine from
adenosine and homocysteine. The fact that human placental SAHH has a
single binding site for copper per 48-kDa subunit that has a K 5.8
10
M is clearly consistent with SAHH being CuBP, i.e. human SAHH has a copper binding activity similar to that of
CuBP(2) . Finally, the fact that mouse liver CuBP has SAHH
catalytic activity confirms that CuBP is SAHH. Incubation of mouse
liver CuBP with 5 molar equivalents of NAD
activated
its hydrolase activity 33-fold. Although the mouse liver enzyme was
still 15-fold less active than the human placental SAHH, CuBP has high
SAHH activity. The hydrolysis reaction catalyzed by SAHH is unusual in
that it requires an NAD
-dependent oxidation step prior
to the hydrolysis step(38) . The reverse reaction, SAH
synthesis, also begins with an oxidation step that requires
NAD
(38) . Thus, this reaction could not be
catalyzed by a nonspecific hydrolase that is not homologous to SAHH.
The fact that CuBP, as isolated, was inactive was not surprising in
view of the SAHH literature. SAHH, as isolated, was often either
inactive or partially active(34) . The chelating Superose step
in the purification of CuBP involves a pH 4.9 buffer at high ionic
strength, and the phenyl Superose step involves isolating CuBP at very
low ionic strength(1, 2) . Either of these steps may
have partially stripped NAD
or NADH from CuBP, thereby
accounting for the partial activation that was detected when
NAD
was added. Further, it was not surprising that
CuBP was not fully activated because the NAD
in SAHH
can become irreversibly reduced to NADH(36, 39) .
Since NAD/NADH is very tightly bound to SAHH, the only method to fully
activate SAHH is to acid precipitate the protein and replace the NADH
that is released at low pH with
NAD
(35, 37) .
A significant role for CuBP/SAHH in cellular copper metabolism is indicated by its high affinity for copper, the fact that SAHH levels are affected by copper levels(2) , and the effects that genetic defects of copper metabolism and copper deficiency have on its properties or levels(2) . The role of CuBP/SAHH in copper metabolism could involve both its enzymatic and copper binding activities. The catalytic activity of SAHH may be related to copper metabolism through its role in cysteine synthesis. SAH is the product of all methyl transferase reactions involving S-adenosylmethionine. SAHH catalyzes the reversible hydrolysis of SAH to adenosine and homocysteine(23) . In vivo, the reaction is driven in the hydrolysis direction by the deamination of adenosine to inosine by adenosine deaminase. In vitro, the synthesis reaction is favored thermodynamically. Homocysteine is a pivotal intermediate for either salvaging methionine or for the synthesis of cysteine. While prokaryotes and plants make methionine from cysteine, the pathway is unidirectional in animals, i.e. animals can only synthesize cysteine from methionine(40, 41) . This is the only pathway for synthesizing cysteine in animals, and cysteine is an essential amino acid in newborns(42) . MT is a cysteine-rich, metal binding (including copper) protein(43) . It was reported that when MT is induced, about 30 percent of the cysteine in a cell is used to synthesize MT(24) . Thus, MT synthesis may link SAHH catalytic activity with copper metabolism. Another possibility involves glutathione (GSH) synthesis. Cysteine is used to synthesize GSH. A direct role for GSH in copper metabolism as a copper trafficking factor has been proposed(44, 45, 46) . Also, the level of reduced GSH may be critical to the formation of Cu(I), which may be essential for copper incorporation into some copper enzymes. More generally, interactions between sulfur metabolism and copper metabolism have not been thoroughly studied and need to be explored further.
Copper is apparently not required for the catalytic
activity of CuBP/SAHH; both mouse liver and human placental CuBP/SAHH
are active as isolated with less than 1 g atom of copper/mol of 48-kDa
subunit(2) . Moreover, copper had no significant effect on
CuBP/SAHH activity when added in vitro. However, this does not
preclude an effect of copper on SAHH activity and its regulation in
vivo or an effect of copper on cycling between NAD and NADH during catalytic turnover. Regardless of how copper
binding affects CuBP/SAHH activity, decreased levels of copper in
copper deficiency most likely decrease SAHH activity by reducing the
steady state levels of CuBP/SAHH protein(2) .
The
concentration of SAHH in the liver and its Kfor copper binding are consistent with a significant role of SAHH
in regulating the distribution of intracellular copper through its
copper binding activity. The affinity of CuBP/SAHH for copper is
similar to that of albumin(47) , which is known to transport
and deliver copper to the liver and other
tissues(48, 49) . Thus, in analogy to copper delivery
from albumin, the K
for copper binding to
CuBP/SAHH indicates that copper bound to CuBP/SAHH could equilibrate
with various copper pools, copper enzymes, or other proteins involved
in copper metabolism. Also, CuBP/SAHH constitutes
0.5% of total
hepatic cytosolic protein(1, 2) . This high
concentration coupled to its high affinity for copper indicates that
CuBP/SAHH is likely to compete well with other cellular copper binding
proteins in the liver, and CuBP/SAHH seems to make a significant
contribution to the total copper binding activity of hepatic
cytosols(1) . Thus, the affinity of CuBP/SAHH for copper is
high enough for it to compete well with other copper binding proteins
in liver cells, yet low enough for CuBP/SAHH to supply its copper to
copper pools, which are active in intracellular copper metabolism, and
any changes in the levels of CuBP/SAHH or its binding affinity for
copper should significantly alter the intracellular distribution of
copper within the liver. SAHH may also bind other metals and play a
role in their distribution.
The results obtained here suggest that CuBP/SAHH is a bifunctional protein with roles in copper metabolism and sulfur amino acid metabolism. Recently, several proteins have been identified as bifunctional proteins. Aconitase, an enzyme in the Krebs cycle, has been shown to be a posttranscriptional regulator of transferrin and ferritin mRNA expression(50) . Glyceraldehyde-3-phosphate dehydrogenase, an enzyme in glycolysis, also binds tRNA and may be involved in nuclear RNA export(51) . As discussed above, CuBP/SAHH may have a bifunctional role in copper metabolism through its enzymatic and copper binding activities. Moreover, the effects of copper on CuBP/SAHH levels may affect methionine and cysteine metabolism under some conditions(2) . In particular, methylation may be affected since SAHH activity is required to remove SAH, which is a potent competitive inhibitor of methyltransferases(52) .