(Received for publication, December 20, 1995; and in revised form, January 16, 1996)
From the
Properties of the L- and H-type polypeptide subunits forming
ferritin 24-mer molecules in mice were investigated, using the products
of in vitro transcription and translation from the two cloned
genes, and recombinant ferritin molecules (HL
or H
L
) produced by transformation in Escherichia coli. Several different conditions for analytical
electrophoresis reproducibly show that the relative migration position
of the two mouse ferritin subunits is reversed from that reported for
ferritin H- and L-subunits in all other mammals; since mouse and human
H-polypeptides almost co-migrate, this unusual relative mobility is due
largely to novel properties of the murine L-subunit.
This unusual electrophoretic property of the mouse L-subunit has led to conflicting reports about the subunit composition of natural mouse ferritin. Here, we show that the single major electrophoretic band given by liver ferritin purified from mice having a short-term iron overload matches that produced by the genetically defined L-polypeptide and that some bona fide H-subunits are also detected.
In conclusion, it is reasonable to assume that, when mouse ferritin samples will be analyzed under the same conditions as those described here, the slower species will correspond to the L-type subunit. However, when dealing with ferritin from species other than human or mouse, it should be kept in mind that upon electrophoretic analysis of ferritin polypeptide, the designation of an electrophoretic band as being H- or L-type subunits will be very uncertain without corroboration from genetic, immunological, or amino acid sequencing data.
Regulated gene expression of several different highly specialized proteins enables eukaryotic cells to achieve homeostasis of their iron content and iron metabolism(1) . The metalloprotein, ferritin, is the major binder of non-heme iron within the cytoplasm of cells in humans, other vertebrates, invertebrates, fungi, and plants, as well as many different bacteria (see (2, 3, 4, 5) for reviews). The ability of ferritin to keep unused intracellular iron in a soluble and relatively safe form balances the essential cellular requirement for this metal with the potential danger of its producing oxidative stress by catalyzing the formation of oxygen free radicals.
Each ferritin
molecule in mammals can bind up to several thousand atoms of ferric
iron as microcrystallites of the biomineral,
ferrihydrite(2, 3) . The special quaternary structure
of this large metalloprotein (diameter = 130 Å (13
nm)) determines that the bound iron is surrounded by an outer shell
made from 24 associated polypeptide
subunits(2, 3, 4, 5) . The two kinds
of ferritin subunits in mammals, H (heavy)-type (molecular mass
=
21 kDa) and L (light)-type polypeptides (molecular mass
=
19 kDa), have over 50% amino acid sequence homology (see (4, 5, 6) for reviews). Both subunits fold
into similar globular units at the secondary and tertiary levels of
protein structure (2) and readily co-assemble in various
proportions to form a family of mostly heteropolymeric molecules (e.g. H
L
,
H
L
, H
L
,
etc.)(7) . Although these two subunits share many structural
features, they now are known to have some different functional
properties (e.g. see (8) and 9).
The two major
kinds of polypeptide subunits in mammalian ferritin and apoferritin
molecules have been defined rigorously at the levels of amino acid
sequence, nucleotide sequence of their genes or cDNAs, and reactivity
with subunit-specific monoclonal
antibodies(6, 10, 11) . However, it is a very
common practice with electrophoretic analysis of ferritin to designate
the more anodal band in the pair of resolved polypeptides as L-type
subunits, and the more cathodal band as H-type
subunits(7, 10) . Many current research studies are
using SDS-PAGE ()analysis of ferritin expression in mouse
cells to investigate eukaryotic gene regulation and cellular iron
physiology. Although the products of in vitro transcription
and translation from cDNAs for mouse ferritin H- and L-polypeptides
revealed that their electrophoretic migration positions were the
opposite of those reported for all other mammalian species (12) , the bands of murine ferritin subunits resolved with
SDS-PAGE continue to be misidentified in many current studies. This
necessarily has resulted in some mistaken and misleading conclusions.
Genes and cDNAs encoding both types of mouse ferritin subunits have been cloned and sequenced in several laboratories(12, 13, 14, 15, 16) . To further analyze the properties of bona fide murine ferritin H- and L-type subunits, the present study has used polypeptides encoded by these cDNAs; the genetically defined polypeptides also have been compared directly to the natural subunits of purified mouse liver ferritin. Our results show that migration of the mouse L-polypeptide is reproducibly slower than that of the mouse H-polypeptide under several different conditions for electrophoresis; the observed inversion of the usual relative positions for ferritin H- and L-subunits with SDS-PAGE is caused by the anomalous properties of the murine L-subunit. Thus, it is risky to assign a given SDS-PAGE band to ferritin H- or L-type subunits in other species without having a confirmation from either genetic, immunological, or amino acid sequence data.
Figure 1: Comparison of mouse and human ferritin polypeptides resolved by SDS-PAGE. Specific immunoprecipitates of newly synthesized ferritin from human lymphoblastoid cells (lanes 1 and 2; lane 1 has twice the sample amount as lane 2) and from mouse erythroleukemia cells (lane 4) were resolved on a 12% SDS-PAGE slab. Lane 3 is a blank lane, containing nonradioactive standards for molecular mass. Products of in vitro transcription and translation from plasmids encoding the mouse ferritin H-subunit (lane 5) or L-subunit (lane 6) were run on a similar gel and aligned with the in vivo synthesized mouse subunits. Respective positions of human (left) and mouse (right) H- and L-subunits are indicated. Anode (+) is below.
Figure 2: Identification of polypeptide subunits in recombinant homopolymeric 24-mer mouse ferritin molecules, using three different protocols for SDS-PAGE. Details about analytical electrophoresis are given under ``Materials and Methods.'' All gels were stained to reveal total protein content. Anode (+) is below. A, SDS-PAGE with separating gel of 15% polyacrylamide. B, SDS-PAGE with separating gel containing 20% polyacrylamide and 10% glycerol. C, SDS-PAGE with separating gel having 15-30% exponential gradient of polyacrylamide. Samples were 24-mer H-homopolymers (lanes 1), a mixture of H-homopolymers and L-homopolymers (lanes 2), and 24-mer L-homopolymers (lanes 3). Relative separation of the two types of ferritin subunits, bandwidth, and resolution of minor bands all vary according to the characteristics of each SDS-PAGE protocol.
Figure 3: Comparison of polypeptide bands resolved by SDS-PAGE in recombinant homopolymeric mouse ferritin molecules, with those in pure natural liver ferritin molecules from mice having iron overload. SDS-PAGE with 15-30% exponential gradient of polyacrylamide, stained to reveal total protein content. Anode (+) is below. Samples are a mixture of pure recombinant H- and L-homopolymers (lane 1), pure mouse liver ferritin (lanes 2 and 4, each from a different gel), molecular mass standards (lane 3), and pure recombinant L-homopolymer (lane 5). The bands shown for the molecular mass standards are: carbonic anhydrase, 29 kDa; trypsinogen, 24 kDa; soybean trypsin inhibitor, 20.1 kDa. Positions of H- and L-subunits are indicated together with the H band in pure liver ferritin (arrowheads) and a slower band common to pure liver ferritin and the recombinant L-homopolymer (arrow).
Use of genetically defined polypeptides permits a rigorous identification of the H- and L-type subunits forming ferritin and apoferritin molecules. The present results clearly show that the more cathodal migration position of the mouse ferritin L-subunit is observed reproducibly with several different systems for SDS-PAGE and therefore is not likely to be some technical artifact; moreover, the unusual migration position of the mouse L-polypeptide is observed whether this is synthesized in vitro from cloned genes, in vivo within cultured cells, or within E. coli as recombinant proteins. The pattern of relative migration of the two ferritin subunits in mice is the opposite of that reported for ferritin subunits in all other mammalian species examined to date, where the L-subunit always migrates to the anodal side of the H-subunit (e.g. humans, horses, and rats(7, 10) ; rabbits(25) ). Since mouse H- and human H-subunits almost co-migrate, the reversed relative positions of H- and L-subunits in mice must be due to novel properties of the murine L-polypeptide.
What causes the unusual migration of the mouse ferritin L-subunit is not clear. Amino acid sequence and composition derived from cloned cDNAs for the two mouse ferritin polypeptides (12, 13, 14, 15) are quite similar to the consensus for each ferritin subunit in humans and other mammals(5, 6, 10, 26, 27, 28) ; despite this similarity, the mouse L-subunit uniquely has an anomalous migration with SDS-PAGE. Although some differences in content of certain residues are recognized for mouse versus human ferritin L-subunits (Table 1), none can be expected to cause the observed change in SDS-PAGE migration since these nonglycosylated polypeptides are reduced and denatured for this analysis. Differences in the primary sequence are expected to influence the SDS-PAGE migration only if the denaturing conditions do not completely remove all elements of secondary structure; such causation for anomalous SDS-PAGE migration of certain polypeptides has been postulated previously for other proteins (e.g. see (29) ).
Each ferritin subunit consists of a bundle of four antiparallel
-helices (A to D) with a fifth helix (E) lying at a right angle to
the main axis(2) . An additional octapeptide is inserted into
the turn between the D and E helices of ferritin L-subunits in mice and
rats (12, 27) and causes both nominal mass and chain
length of the H- and L-polypeptides to be nearly equal (Table 1).
However, here we demonstrate that the electrophoretic mobility of mouse
H- and L-polypeptides are clearly different. This condition is mostly
due to the unusual mobility of the L-subunit, which is intermediate
between the 20.1- and the 24-kDa molecular mass markers (see Fig. 3) and slower than would be expected from its calculated
molecular weight (M
= 20,802). It is
possible that the extra octapeptide, although not altering the
three-dimensional structure of the ferritin L-subunit(30) ,
might affect its electrophoretic mobility.
It is difficult to evaluate from the available literature whether the rat L-subunit presents the same unusual electrophoretic mobility. There is a 92% homology between the amino acid sequence for the ferritin L-subunits of rats (27) and mice(12) . However, the additional octapeptide specific of rodent L-subunits differs at three positions between rat (QTGVAQAS) and mice (QTGAPQGS) and might not alter the electrophoretic properties of each L-polypeptide to the same extent. Therefore it seems worthwhile to reevaluate the electrophoretic properties of the polypeptide products encoded by the cloned genes for rat ferritin subunits.
The subunit composition of natural mouse
ferritin molecules has remained quite controversial (17, 31) due to the unusual reversal of the
traditional positions for H- and L-subunits with SDS-PAGE. The present
analysis of genetically defined polypeptides now demonstrates that most
subunits in liver ferritin molecules from mice with parenteral iron
overload are L-type polypeptides. Two minor bands originally were
resolved with SDS-PAGE of this natural ferritin(17) . One of
these minor bands (Fig. 3, arrow) migrates to the
cathodal side of the single major band and commonly would be identified
as an ``H-subunit''; our present results clearly show that it
corresponds only to the L-subunit. However, bona fide H-type subunits
now also have been detected in small amounts within this natural liver
ferritin, indicating that these mouse ferritin molecules indeed are at
least partly heteropolymeric (e.g. HL
).
An increasing number of research investigations using murine cells to study gene regulation and iron metabolism are reporting the designation of H- or L-type subunits for SDS-PAGE bands derived from anti-ferritin immunoprecipitates. Based upon our results, many of these published identifications are incorrect and the deduced conclusions therefore are misleading. We hope that the present data will prevent further mistaken identifications from being made.
Note Added in Proof-We have just become aware that the genetically-defined rat ferritin L-polypeptide has been shown to migrate to the anodal side of the rat H-polypeptide(32) . This result indicates that 1) the anomalous relative migration of ferritin L-subunits from mice is not found with those from the other rodent, and 2) the novel migration of the mouse L-subunit is not due simply to the presence of eight additional amino acids (since an octameric peptide also is inserted into rat L-subunits).