(Received for publication, November 1, 1995; and in revised form, January 26, 1996)
From the
The deep-sea tube worm Riftia pachyptila Jones
possesses a complex of three extracellular Hbs: two in the vascular
compartment, V1 (3500 kDa) and V2 (
400 kDa), and one in the
coelomic cavity, C1 (
400 kDa). These native Hbs, their
dissociation products and derivatives were subjected to electrospray
ionization mass spectrometry (ESI-MS). The data were analyzed by the
maximum entropy deconvolution system. We identified three groups of
peaks for V1 Hb, at
16, 23-27, and 30 kDa, corresponding to
(i) two monomeric globin chains, b (M
16,133.5)
and c (M
16,805.9); (ii) four linker subunits,
L1-L4 (M
23,505.2, 23,851.4, 26,342.4, and
27,425.8, respectively); and (iii) one disulfide-bonded dimer D1 (M
31,720.7) composed of globin chains d (M
15,578.5) and e (M
16,148.3). V2 and C1 Hbs had no linkers and contained a glycosylated
monomeric globin chain, a (M
15,933.4) and a
second dimer D2 (M
32,511.7) composed of chains e
and f (M
16,368.1). The dimer D1 was absent from
C1 Hb, clearly differentiating V2 and C1 Hbs. These Hbs were also
subjected to SDS-PAGE analysis for comparative purposes. The following
models are proposed ((cD1)(bD1)
) for the one-twelfth
protomer of V1 Hb, ((cD)(bD)
(aD)) (D corresponding to
either D1 or D2) for V2 and C1 Hbs. HBL V1 Hb would be composed of 180
polypeptide chains with 144 globin chains and 36 linker chains, each
twelfth being in contact with three linker subunits, providing a total
molecular mass = 3285 kDa. V2 and C1 would be composed of 24
globin chains providing a total molecular mass = 403 kDa and 406
kDa, respectively. These results are in excellent agreement with
experimental M
determined by STEM mass mapping and
MALLS(8) .
Several models have been proposed for the quaternary structure
of annelid hexagonal bilayer hemoglobins (Hbs)()(1, 2, 3, 4, 5) and
vestimentifera Hbs (6) but no definitive agreement exists.
However, it is well established that HBL Hbs have a hexagonal symmetry
and consist of two types of chains, globin-chains (
17,000 Da)
accounting for approximately 70% of total mass and heme-deficient
linker chains (
24,000-32,000 Da) necessary for assemblage
into the HBL structure(7) . A detailed understanding of this
molecular structure requires the knowledge of: (i) the molecular mass
of the native molecule; (ii) the number and proportions of all
constitutive polypeptide chains; (iii) the number and relation between
subunits; and (iv) the determination of linker proportions.
In a
companion study (8) we have confirmed that Riftia
pachyptila (Jones), a vestimentiferan living around deep-sea
hydrothermal vents(9, 10, 11) , possesses
three hemoglobins, two of them dissolved in the vascular blood (V1 and
V2), and one in the coelomic fluid (C1). Their molecular weights have
been determined by scanning transmission electron microscopy mass
mapping (STEM) and by multi-angle laser light scattering (MALLS). Both
methods yielded approximately the same molecular weights with mass
significantly higher than the literature data for V1. V1, V2, and C1
had M of 3396 ± 540
10
,
393 ± 71
10
, and 410 ± 51
10
by STEM, and 3503 ± 13
10
,
433 ± 8
10
, and 380 ± 4
10
by MALLS, respectively. However, STEM mass mapping
measurements for V1 Hb were actually made on one-twelfth molecules (8) and the M
noted above is therefore an
approximation, probably less accurate than MALLS result. Transmission
electron micrographs of V1 are typical of an hexagonal bilayer
hemoglobin (HBL Hb).
Reduced annelid Hbs exhibit a variety of
SDS-PAGE profiles, ranging from one subunit of M 13,000-15,000 to seven subunits in the range of M
13,000 to 40,000 (12) . More recently, a
more accurate technique, ESI-MS, has been successfully applied to some
HBL Hbs allowing the determination of the complete polypeptide chain
compositions of these Hbs(13, 14, 15) . They
consist of 4 to 6 globin chains in the range 16,000-18,000 Da and
3-4 linker chains in the range 24,000-27,000 Da. Previous
studies by SDS-PAGE on Riftia vascular Hbs have shown that
they consist of subunits of M
15,000, 30,000,
and 45,000 (16, 17, 18) . The amino acid
compositions of these Hbs are very similar to each other and they
contain 1 heme per 23,000 g of protein as in annelid Hbs(17) .
The aim of the present work was to conceive realistic models of the quaternary structure of Riftia heteromultimeric Hbs. In a companion study (8) we have determined the precise masses of three Riftia Hbs. In this report we have determined the stoichiometry of the polypeptide chains and subunits constituting these Hbs by ESI-MS coupled with maximum entropy(19) . In addition, these results are compared with those obtained by SDS-PAGE.
Figure 1:
SDS-PAGE profiles of R. pachyptila Hbs V1 (lanes 1 and 2), V2 (lanes 3 and 4) and C1 (lanes 5 and 6). Lanes 1, 3, and 5, unreduced Hb; lanes 2, 4,
and 6, reduced Hb. Relative molecular weights (M) were estimated using low molecular weight
protein calibrants (Pharmacia).
V2 and C1 have similar
dissociation patterns with three subunits of M 16000, 19,000 and 29,000: B1, B2, and B3 (lane 3), and
C1, C2, and C3 (lane 5), respectively. Upon reduction V2 and
C1 dissociate into four polypeptide chains with M
ranging from 16,000 to 19,000: BI to BIV (lane 4) and CI
to CIV (lane 6), respectively. Subunits B3 and C3, like
subunit A3 in V1 Hb, correspond to disulfide-bonded dimers of globin
chains BI and BII, and CI and CII, respectively.
Figure 2: ESI-MS spectra of R. pachyptila native Hbs. A, raw spectrum of Hb V1, consisting of a series of multiply charged ions from each component on a mass-to-charge (m/z) ratio scale, m/z = (M + nH)/n, where M is the mass of the component, H is the mass of the proton and n is a series of consecutive integers. B-D, MaxEnt processed spectra of Hbs V1, V2, and C1 presented on a true mass scale.
The mass spectrum of V2 Hb (Fig. 2C) consisted of
two groups of components, one around M 16,000-18,000 and the other over 30,000. The first group
(close-up in Fig. 3A), in addition to chain b and c,
contained five glycosylated isoforms of chain a (a1, M
17,636.4 ± 1.0, a2, M
17,799.0
± 1.0; a3, M
= 17,962.3 ±
0.1; a4, M
18,451.0 ± 1.1; a5, M
18,613.2 ± 0.5) which could be identified
by comparison with the mass spectrum of the deglycosylated V2 Hb (Fig. 3B). It should be noted that deglycosylation of
V1 Hb did not bring any change in the spectrum. The second group
contained D1 and another dimer D2 = 32,511.7 ± 1.3 (Fig. 2C), which after reduction appeared to be
constituted of chains e and f, M
16,368.1 ±
0.3 (Fig. 3C). The mass spectrum of C1 Hb (Fig. 2D) resembles that of V2, except that it does not
contain dimer D1. Table 1lists the average masses of the various
components of V1, V2 and C1 Hbs. These results clearly revealed that
(i) HBL Hb V1 was devoid of glycoprotein and possessed linker chains
(L1-L4), (ii) V2 and C1 Hbs possessed glycosylated chains but no
linker chain, and (iii) V2 and C1 were different Hbs, the former
containing a dimer absent from the latter.
Figure 3: MaxEnt processed ESI-MS of various derivatives of R. pachyptila Hb V2 presented on an expanded mass scale. A, native; B, deglycosylated; C, reduced; and D, reduced and carbamidomethylated.
We have also analyzed the dissociation products V3, V4 and C2(8) . V3 consisted of the dimer D1, while V4 was composed of the monomers b, c and the glycosylated isoforms of chain a. The mass spectrum of C2 was identical to that of C1.
Carbohydrate and Cysteine Residue Contents- Fig. 3presents the mass spectra of Hb V2 after various
treatments. Deglycosylation allowed us to determine the mass of chain a (M 15,933.4 ± 0.3; Fig. 3B)
and we found that the glycosylated isoforms a1-a5 differ in mass
by one or more hexose residue(s) (162.1 Da). The difference in mass
between chain a5 and chain a (2680.3 Da) is, within experimental error,
the mass of the glycan residue (HexNAc)
(Hex)
(2676.40 Da). Note that ESI-MS cannot distinguish between
monosaccharide residues because they have identical masses.
Comparing mass spectra obtained after carbamidomethylation with or
without reduction, we have determined the numbers of free Cys residues
and disulfide bonds. Fig. 3D shows the results obtained
after reduction and carbamidomethylation of V2 Hb. Monomeric chains b
and c, and the different glycosylated isoforms of chain a possess one
intrachain disulfide bond. Chain b contains also one free Cys residue.
Dimers D1 and D2 contain only one interchain disulfide bond which is
quite resistant to reduction since 50% of the dimers are still
present after 60 min with 5 mM DTT (data not shown). Each
chain composing D1 and D2 (d, e, and f) contains one intrachain
disulfide bond but only chain e has an additional free Cys. We have
also found twelve Cys residues on the linker chain L1. Table 2summarizes glycan composition and number of Cys residues
for all chains.
MaxEnt analysis of ESI-MS spectra produces quantitative relative
intensity data. It provides a zero charge spectrum in which the areas
under the peaks are proportional to the sum of the intensities of the
peaks in the original raw multicharged spectrum. Using mass
measurements of the different Hbs obtained by MALLS (8) and the
exact masses of polypeptide chains determined by ESI-MS, we propose
coherent models of the quaternary structure of Riftia Hbs.
According to these results HBL Hb V1 would consist of 144 globin-like
chains (48 monomers and 48 dimers) and 36 linker chains (Table 3). In this model, the relative intensities of the four
linker chains were L1:L2:L3:L4 = 1:1:0.5:0.5, the globin:linker
ratio was 0.75:0.25 and the mass of protein per heme yielded 22,805 g.
The primary functional subunit of HBL Hb V1 corresponds to a dodecamer
((cD1)(bD1)) with a calculated molecular mass of 200 kDa.
This basic subunit should be bound to three linker chains (e.g. 1 L1, 1 L2, and either 1 L3 or L4) corresponding to a total linker
mass of 74 kDa. Finally we obtain for the whole molecule a molecular
mass of 3285 kDa.
In the same way, Hb V2 (Table 4) and C1 (Table 5) would be constituted of 24 globin-like chains providing
a calculated mass of 403 and 406 kDa including heme and glycan side
chains, respectively. We propose the following molecular models,
((cD)(bD)(aD)) for Hb V2 (D being D1 or D2) and
((cD2)(bD2)
(aD2)) for Hb C1. The dissociation product V3 is
composed of ((D1)
) (99 kDa, calculated mass, Table 6). The dissociation product C2 is composed of
((cD2)(bD2)
(aD2)) (206 kDa, calculated mass, Table 7).
SDS-PAGE has been widely used in the past to study the structure of proteins, including HBL Hbs of annelids (12) and vestimentifera(6, 29) . Even though ESI-MS gives more accurate results (see below) very few HBL Hbs have been studied with this technique to date (13, 14, 15) and thus we continued using SDS-PAGE for comparative purposes.
Our SDS-PAGE results on Riftia Hbs are in total agreement with those obtained on Lamellibrachia sp. Hbs (6) and clearly reveal that only HBL Hb V1 possesses linker chains with molecular mass of 32 and 36 kDa (AV-AVIII). Moreover, upon reduction these polypeptide chains display bands with a reduced mobility, indicative of even higher molecular masses. This phenomenon has been previously observed in other HBL Hbs (6, 12) and is probably due to the presence of intrachain disulfide bonds(12) . It is known that unreduced proteins containing intrachain disulfide bonds possess a more compact hydrodynamic shape in SDS than the fully reduced proteins (30, 31) . The primary structure of some linker chains of Lamellibrachia sp.(32) , Tylorrhynchus heterochaetus(33) , and L. terrestris(34) revealed an important number of cysteine residues which may be able to form intrachain disulfide bonds. In addition, SDS-PAGE overestimates the true molecular mass of components containing glycan or a high content of some amino acids such as proline(35, 36) .
In contrast, ESI-MS analysis of Riftia Hbs provides a complete and self-consistent description
of their constituent subunits and polypeptide chains, together with
mass measurements with an unrivaled accuracy (0.01%). Moreover, the
high resolution (1500) allows polypeptide chains with close
molecular masses to be distinguished. Vascular Hb V1 consists of two
monomeric globin chains b and c, one disulfide-bonded dimer (D1) of
globin chains d and e, and four different linker chains L1-L4.
Vascular Hb V2 consists of three monomeric globin chains, a, b, and c,
chain a occurring as five glycosylated forms (a1-a5), and two
disulfide-bonded dimers, D1 and D2, the latter consisting of chains e
and f. These results imply that V2 is not a subunit of V1. Coelomic Hb
C1 shares with the vascular Hb V2, the three monomeric globin chains a,
b, and c, including the glycosylated isoforms a1-a5, the
disulfide-bonded dimer D2 and the absence of linker subunits. However,
it lacks the dimer D1 and therefore, in contrast with previous
studies(16) , our results show that V2 and C1 are different
Hbs.
Comparing our results with those obtained with this technique
on the earthworm, Lumbricus(13) , and the leech Macrobdella(14) HBL Hbs, it is clear that all three
Hbs are built up from the same kind of constituents, e.g.
monomeric globin chains (16 kDa), linker chains (
26 kDa), and
trimers or dimers of globin chains (
53 or 32 kDa, respectively).
In Riftia, the linker chains L1-L4 are only found in V1
Hb, in agreement with the idea that they are required for the
maintenance of the HBL structure by analogy with annelid HBL Hbs, such
as earthworm Lumbricus(13) and the leech Macrobdella(37) , and annelid chlorocruroin, such as
the polychaete Eudistylia(38) . Riftia and Lamellibrachia(6) HBL Hbs most closely resemble that
of the achaete Macrobdella Hb(14) . These Hbs contain
disulfide-bonded dimers whereas oligochaetes and polychaetes Hbs
usually contain disulfide-bonded trimers(12) . However, in
contrast with annelid species, the vestimentifera Riftia pachyptila and Lamellibrachia sp. (29) possess an original
multi-hemoglobin system with two extracellular Hbs (V2 and C1) in
addition to the HBL Hb V1.
ESI-MS also offers reliable information concerning the number of Cys residues and disulfide bonds in native proteins, by comparing data obtained from native or reduced forms with their carbamidomethylated counterparts (see e.g.Fig. 3, C and D). All annelid and tube worm globin-like chains known to date can be classified into four groups. All groups possess an intrachain disulfide bond, and three of these groups contain a supplementary free Cys, at different positions, participating in an interchain disulfide bond(29, 39) . In Riftia Hbs (Table 2), all chains have at least two Cys, forming an intrachain bond. Chains d, e, and f have an additional Cys forming an interchain bond, but only chains b and e have a free Cys residue. We have been able to analyze only one linker chain (L1) and we found a considerable number of Cys residues, all participating in intrachain bonds. The knowledge of Cys contents is not only important for structural implications, but, in the case of vestimentifera, it may also have a functional meaning since previous studies have suggested a role for Cys in the unique ability of these Hbs to reversibly bind sulfide(6, 16, 32) .
Using the precise masses of native Riftia Hbs (8) and the present results on polypeptide chain composition, we have been able to propose coherent models of the quaternary structure of these heteromultimeric proteins. Data derived from SDS-PAGE analysis of V2 and C1 Hbs yields a 28-chain model in total agreement with the model proposed for Lamellibrachia sp.(6) . Using data derived from ESI-MS results we found a slightly different model composed of 24 globin chains, suggesting an association of 8 trimeric subunits each composed of one disulfide-bonded dimer and a monomeric chain. The total calculated mass of this model, including heme groups and glycan side chains, is very close to the experimental mass (see Table 4and Table 5).
The model found for V1 Hb (Table 3) consist of
144 globin chains and 36 linker chains, providing a total calculated
mass of 3285 kDa, including heme groups. This is very close to the
experimental data obtained by STEM mass mapping (3396 ± 540
10
) and MALLS (3503 ± 13
10
)(8) . In addition, this model yields a heme
content consistent with experimental data found
previously(16) . The primary functional subunit of HBL Hb V1
corresponds to a dodecamer with a molecular mass of 200 kDa and should
be linked with three linker chains corresponding to a mass of 74 kDa.
This assemblage provides a molecular mass of 274 kDa, in good agreement
with the one-twelfth mass of 283 kDa determined by STEM mass mapping (8) . As for other HBL Hbs analyzed with ESI-MS to
date(13, 14, 15) , our data for Riftia HBL Hb V1 fit well with the ``bracelet model'' proposed
for L. terrestris(40) . This model has recently been
substantiated by three-dimensional reconstruction based on
cryomicroscopy analysis(41) .
The results obtained with Riftia Hbs illustrates the power and scope of ESI-MS in the analysis of large, heteromultimeric protein complexes. The precise knowledge of the quaternary structure of Riftia Hbs will certainly have consequences in functional and evolutionary studies.