Spectrin’s chimeric E2/E3 enzymatic activity

Abstract

In this minireview, we cover the discovery of the human erythrocyte α spectrin E2/E3 ubiquitin conjugating/ligating enzymatic activity and the specific cysteines involved. We then discuss the consequences when this activity is partially inhibited in sickle cell disease and the possibility that the same attenuation is occurring in multiple organ dysfunction syndrome. We finish by discussing the reasons for believing that nonerythroid α spectrin isoforms (I and II) also have this activity and the importance of testing this hypothesis. If correct, this would suggest that the nonerythroid spectrin isoforms play a major role in protein ubiquitination in all cell types. This would open new fields in experimental biology focused on uncovering the impact that this enzymatic activity has upon protein–protein interactions, protein turnover, cellular signaling, and many other functions impacted by spectrin, including DNA repair.

The erythrocyte spectrin membrane skeleton

The erythrocyte, or red blood cell (RBC), travels the circulatory system for 120 days in people who are not anemic. During this four-month journey, this 8 µm, biconcave disc must pass through the circulatory system, which narrows to 2 µm in the smallest venules. To accomplish this feat, the RBC must be reversibly deformable and elastic, but at the same time have membrane properties that maintain the cell’s structural integrity despite the shear forces that it encounters. The spectrin membrane skeleton is a two-dimensional meshwork of proteins that spans the cytoplasmic membrane surface of the RBC, and provides it with these properties as well as maintaining its biconcave shape (Figure 1). A sampling of important prior reviews is provided.^1–6

Figure 1.

The RBC spectrin membrane skeleton. The RBC is a biconcave disc (upper left) which on its cytoplasmic surface (light blue) contains a network called the spectrin membrane skeleton. The detailed protein interactions constituting this membrane skeleton are shown in the center. In the upper right-hand corner is a diagram showing that actin assemblies, including protein 4.1 or adducin troponin and tropomodulin, form a hexameric array that is crosslinked by spectrin tetramers. The drawing below gives a transbilayer view of the protein assembly, which attaches spectrin, via ankyrin, to the band 3 tetramer (with associated 4.2 and GLA).

⊥αSp: alpha spectrin; βSp: beta spectrin; B3: band 3; Ank: ankyrin; TMOD: tropomodulin; TN: troponin; GLA: glycophorin A; 4.1: protein 4.1; GLC: glycophorin C; and 4.2: protein 4.2

The membrane skeleton, visualized by negative staining and electron microscopy, is primarily a hexagonal lattice. The lattice contains central actin protofilaments interconnected by spectrin tetramers.⁷ Spectrin in its simplest form is an antiparallel αβ heterodimer, which in vivo forms an (αβ)₂ tetramer by head-to-head linkage of two heterodimers.^8,9 The α and β spectrin subunits contain a primary repeating unit of ∼106 amino acids called the spectrin repeat. Complete amino acid sequence deduced for the spectrin subunits demonstrated multiple spectrin repeats in both the α (∼20) and β (∼16), which are numbered beginning at the N terminus by convention.^10–12 Karinch et al.¹³ demonstrated that the tail ends of spectrin tetramers, which do not contain the spectrin repeats, attach to actin protofilaments associating with residues 47 through 186 at the N-terminus of the β subunit. To reinforce this spectrin–actin interaction, protein 4.1 binds to the tails of β spectrin creating a spectrin–4.1–f-actin ternary complex.^14–17 Another peripheral membrane protein named adducin binds to both β spectrin and the barbed fast-growing end of F-actin.^18–20 Attachment of the membrane skeleton to the membrane occurs in at least two ways. Protein 4.1 that binds to β spectrin, close to the N-terminal actin-binding domain also binds glycophorin C.^21,22 Ankyrin binds β spectrin^15,23 to the anion transport channel, Band 3.^24,25

Discovery of spectrin’s chimeric E2/E3 ubiquitin conjugating/ligating activity

From its earliest description in 1968,²⁶ to its appreciation as a major component of the membrane skeleton in the early 1980s,¹ spectrin was thought to play the structural role as indicated in Figure 1. In 2001, the understanding of spectrin function was extended when Kakhniashvili et al.²⁷ demonstrated that spectrin also had an enzymatic activity that allowed it to ubiquitinate itself. The story of this discovery was preceded by a bit of serendipity. A rabbit autoantibody stained control RBC membrane skeletons brightly, but showed very weak staining when indirect immunoflourescence (IF) was performed on homozygous (SS) sickle cell anemia (SCA) RBC membrane skeletons. Interestingly, when running sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of RBC membranes, without reducing agent, a prominent band was observed above α spectrin in the case of control RBCs, which was substantially diminished in RBC membranes derived from a patient with SCA (but not a person with sickle cell trait). This band of interest was stained by the autoantibody and anti-α-spectrin (but not anti-β-spectrin) antibody on Western blots. Thus, it was named it α^/ spectrin. When SDS PAGE was run in the presence of reducing agent, the α^/ spectrin was diminished in the Coomassie blue-stained gel and the western blots. Now we had an interesting mystery. What was this dithiothreitol (DTT) reducing agent sensitive α′ spectrin?

The journey from this unexplained finding, to an understanding of spectrin’s enzymatic function, began with an article by Kakhniashvili et al.²⁷ In this study, it was first demonstrated that the modification was ubiquitin. Next was the demonstration that erythrocyte α-spectrin is an E2 ubiquitin-conjugating enzyme that is able to target itself.²⁷ The DTT-sensitive adduct, α^/ spectrin, was ubiquitin linked to α-spectrin via a thioester bond. E1 ubiquitin-activating enzyme and ATP were required to form both the α-spectrin–ubiquitin adduct and conjugate. Using computer programs (COMPARE and PEPTIDE STRUCTURE) and a structural prediction program (PROPSEARCH), we analyzed the α-spectrin sequence.²⁷ Analyses indicated that a segment within α-spectrin repeat 20 could be responsible for E2 ubiquitin-conjugating activity. Cysteine residue 2071 was surrounded by a sequence that shared ∼70% identity (11 of 16 residues) with the active site consensus sequence critical for all known E2 enzymes. It had not yet determined whether an additional E3 ubiquitin-ligating enzyme was necessary for the ubiquitination involving α spectrin, or alternatively that spectrin had both E2 and E3 activity. However, a potential E3 site that conformed to the cleft structure surrounding the active site residues of E3 HECT domain enzymes was identified with this analysis. This potential E3 site surrounded Cys2100, which was also located in α-spectrin repeat 20. Furthermore, there was a region of sequence that contained a lysine cluster within repeat 21 (2199-KRKQKEIQAMK-2209) that could possibly contain a lysine acceptor site(s) for ubiquitin attachment.²⁷ Therefore, Kakhniashvili et al. proposed an initial hypothesis where ubiquitin was transferred from an E1 enzyme to cysteine 2071, then from this cysteine to cysteine 2100, and then finally to the lysine-rich region of α spectrin repeat 21.^27,28 This hypothesis was subsequently proven to be partially correct, as we discuss below.

The hypothesis could be tested utilizing a recombinant peptide representing residue 2005 to the C-terminus of α spectrin. Hsu et al.²⁹ cloned this segment into a glutathione S-transferase (GST) vector and demonstrated that this C-terminal α spectrin recombinant had the ubiquitin-conjugating and ligating activity and could transfer ubiquitin to itself. This finding proved that the activity was inherent in the α spectrin structure, rather than a copurifying RBC protein. By testing the C-terminal recombinant protein, GST-fusion α spectrin (2005-2415), using an in vitro ubiquitination assay, these studies demonstrated that both cysteine 2071 and cysteine 2100 are capable of receiving ubiquitin from an E1-activating enzyme and directly transferring ubiquitin to a target lysine within this α-spectrin C-terminal recombinant peptide. Site-specific mutational analyses using the GST-fusion C-terminal human α-spectrin recombinant was employed to examine this activity. Wild-type recombinant protein, α-spectrin (2005–2415) has six cysteines. Mut1 to Mut13 had different combinations of cysteine(s) mutated into alanine(s) (Table 1). Single mutations, such as C2071A or C2100A, were created and the spectrin ubiquitination activity was unaffected by these mutants. Only recombinant peptides containing the C2071A/C2100A double mutant demonstrated loss of activity (Table 1).^29,30 Based on these results, the model shown in Figure 2^29,30 was proposed. In this model, cysteines 2071 and 2100 both have chimeric E2/E3 activity in human RBCs. In mice, and species other than human, residue 2100 is converted to a tyrosine or glutamine suggesting that the conserved cysteine 2071 is the primary E2/E3 site and cysteine 2100 serves a back up function in humans.^29,30

Table 1.

The use of mutational analysis of recombinant peptides representing the C-terminus of α spectrin to define the critical chimeric E2/E3 active site cysteines

Clone	Amino acids						Activity
WT	C2071,	C2100,	C2158,	C2387,	C2298,	C2058	+
Mut1	C2071A,	C2100,	C2158,	C2387,	C2298,	C2058	+
Mut2	C2071A,	C2100A,	C2158,	C2387,	C2298,	C2058	−
Mut3	C2071A,	C2100A,	C2158A,	C2387,	C2298,	C2058	−
Mut4	C2071A,	C2100A,	C2158A,	C2387A,	C2298,	C2058	−
Mut5	C2071A,	C2100A,	C2158A,	C2387A,	C2298A,	C2058	−
Mut6	C2071A,	C2100A,	C2158A,	C2387A,	C2298A,	C2058A	−
Mut7	C2071,	C2100A,	C2158,	C2387,	C2298,	C2058	+
Mut8	C2071,	C2100,	C2158,	C2387,	C2298,	C2058A	+
Mut9	C2071,	C2100,	C2158A,	C2387,	C2298,	C2058	+
Mut10	C2071A,	C2100,	C2158,	C2387,	C2298,	C2058A	+
Mut11	C2071A,	C2100,	C2158A,	C2387,	C2298,	C2058	+
Mut12	C2071,	C2100A,	C2158,	C2387,	C2298,	C2058A	+
Mut13	C2071,	C2100A,	C2158A,	C2387,	C2298,	C2058	+

Source: Modified from Hsu et al.²⁹

Figure 2.

Model of human α-spectrin (2005–2415) ubiquitination enzymatic sites and acceptor sites. Human α spectrin cysteines 2071 and 2100 can both accept ubuiquitin from an E1-activating enzyme and transfer it directly to cysteine(s) with the a21 repeat unit

In summary, Goodman and colleagues had demonstrated that RBC spectrin has a chimeric E2/E3 ubiquitin-conjugating/ligating activity, which is capable of ubiquitinating itself. Later it was shown that it could also ubiquitinate other membrane skeleton-associated target proteins.^31,32 The other known target proteins were ankyrin, protein 4.1, protein 4.2, the anion transport channel, and a protein of currently unknown function (gi 13278939).^31,32

Previously analyzed proteins that also fell into this chimeric E2/E3 category were E2-230K³³ and BRUCE.³⁴ E2-230K is required for remodeling of erythroid cells during differentiation;³³ and BRUCE is a 528-KDa endomembrane-associated protein.³⁴ Corsi et al.³⁵ were the first to demonstrate that RBC spectrin was ubiquitinated and Galluzzi et al.³⁶ demonstrated two ubiquitination sites within α spectrin. The two sites were localized to α spectrin repeat 17 and repeats 20/21 (in agreement with our findings). The lysine involved in ubiquitin linkage to α spectrin repeat 17 is residue 1709.³⁶ There is no currently known function for the repeat 17 ubiquitination site.

Role of the spectrin chimeric E2/E3 activity in sickle cell disease

SCA is the major form of hemoglobinopathies, which result in RBC sickling. This family of diseases is collectively called sickle cell disease (SCD), and SCA was the first described, affects the most people, and is the most severe. The hallmarks of SCA are anemia, vasoocclusion (leading to sickle cell crises), oxidative stress, inflammation, organ damage, and, in many cases, a shortened life span. Within the circulation of a SCA patient, most of the RBCs can convert back and forth from the sickle to the biconcave shape depending on whether hemoglobin-S (HbS) is deoxygenated, with formation of 14 stranded polymers, or oxygenated where the polymers are converted back to HbS monomers. These are referred to as reversibly sickled cells (RSCs). There are other RBCs that are irreversibly sickled cells (ISCs), and highly elongated and dehydrated. The ISCs remain sickled even when HbS is well oxygenated and depolymerized, and account for 2–40% of the RBCs in the circulation of a SCA patient. Previous reviews on the topic will expand upon this discussion.^3,37,38 For much of the 20th century, the molecular basis of ISC formation eluded the research community.

Lux et al.³⁹ demonstrated that most RBC membranes and triton skeletons isolated from ISCs remained sickled. This was not the case for RSC and control membranes and triton skeletons. We defined the molecular defects, within the membrane skeleton, which cause the ISC to be “locked” into an irreversibly sickled shape.^40–46 Shartava et al. demonstrated that core skeletons isolated from ISCs dissociate more slowly than skeletons derived from RSC or control RBCs.^40,41 We then demonstrated that spectrin–4.1–actin ternary complexes, created in vitro from proteins isolated from SCA ISCs verus control RBCs, also dissociate more slowly at 37°C.⁴⁰ Shartava et al.⁴⁰ demonstrated that β actin and spectrin were responsible for the slow dissociation. The defect in ISC β-actin, due to increased oxidative stress, is a disulfide bridge between Cys 284 and Cys 373, which is found at very low levels in RSC and control β-actin.^40,41,44 This post-translational modification in ISC β-actin caused slower and incomplete depolymerization than observed with RSC and control β-actin.⁴³ It did not impact ISC β-actin binding to spectrin.⁴³

SCA RBC α spectrin demonstrates diminished ubiquitination (50–90% reduced) when compared to control RBC spectrin.^28,45,47 This is caused by diminished spectrin E2/E3 activity, which we believe to be caused by the conversion of active site cysteine thiolates (C2071 and C2100) into cysteine oxiforms, which cause loss of function.⁴⁷ As nonubiquitinated α spectrin participates in a more tightly associated spectrin–4.1–actin and spectrin–adducin–actin ternary complex than ubiquitinated spectrin,^45,46 the rate of SCA ternary complex dissociation is far slower than the control ternary complex dissociation rate.⁴⁰ Therefore, the molecular basis of the formation of the ISC is a membrane skeleton that disassembles and reassembles slowly leading to a cell “locked” into the sickled shape. These experiments therefore defined the molecular basis of the ISC being a disulfide bridge in β-actin and diminished ubiquitination of a spectrin, both contributing to a “locked” membrane skeleton and cell (Figure 3).^37,38

Figure 3.

The molecular basis of the ISC. Taken from Goodman³⁷ with permission of the publisher

In SCA patients, there are circulating RSCs and dense ISCs, and both play roles in vasoocclusion.^37,38 We have demonstrated that n-acetyl cysteine (NAC) blocks the formation of ISCs in vitro.^48–50 NACs inhibition of ISC formation correlated with reduction of the ISC β-actin disulfide bridge.⁴⁸ Pace et al. performed a phase II human trial to determine the efficacy of NAC in reducing dense cell and ISC levels, increasing intracellular GSH, and reducing acute vasoocclusive crises (VOC) episodes in SCD patients. At 2400 mg per day, NAC reduced dense cells significantly by 37%, doubled intracellular GSH, and lowered the relative risk of VOC episodes by 61%.⁵¹ Percentage ISCs showed a downward trend at all doses tested (600, 1200, and 2400 mg). The findings of our phase II clinical trial have been confirmed by Nur et al.,⁵² and phase III trials are anticipated.

Erythrocyte spectrin ubiquitination and multiple organ dysfunction syndrome (MODS)

Caprio et al.⁵³ recognized the potential relationship between the role of diminished α^/ or ubiquitinated α spectrin in ISC formation and the rigidity observed in RBCs in MODS. MODS is a major potential clinical problem faced postsurgery. They tested this relationship and demonstrated that the decreased RBC deformability observed in a rat model of trauma and hemorrhagic shock was correlated with diminished α^//α spectrin ratio.⁵³ They concluded that “the fact that α-spectrin, in addition to being a structural membrane protein, has important enzymatic activity that helps regulate the association of the various RBC membrane proteins to the actin cytoskeleton makes it an attractive target for further study and possible therapy” related to MODS.⁵³ We would suggest that this may be true for many disorders in which RBCs are less deformable and misshapen.

Spectrin isoforms in nonerythroid cells

In 1981, Goodman et al.⁵⁴ demonstrated that spectrin and spectrin-related proteins could be found in nonerythroid cells and tissues. This article led to a flurry of articles in the 1980s demonstrating the ubiquity of spectrin and close relatives in mammalian and avian cells.^1,55–62 It became appreciated that spectrin was widely found in eukaryotic cells. Much attention was focused on brain spectrin, because of its very high content in neural tissue (2–3% of the total protein content) and the intrinsic interest in uncovering its neural functions. A large number of comprehensive review articles are available on brain spectrin structure, function, and location,^63–67 which will be briefly covered here. Riederer et al. made the important discovery of multiple isoforms of spectrin in neurons.^68,69 We described an erythroid isoform composed of α subunits, identical to RBC α spectrin, associated with β subunits, which were an alternately spliced forms of RBC β-spectrin, with an extended C-terminus.^68–70 This form, originally called brain spectrin 240/235E and now called αSpI/βSpIΣ2, was found in the soma, dendrites, and postsynaptic densities of all neurons.^68–70 There was a second major form of spectrin in neurons, which had α and β subunits, which were distinct gene products from RBC spectrin but shared ∼60% sequence identity with the RBC spectrin subunits, increasing in sequence identity within the functional domains.^68–70 This isoform, originally called brain spectrin (240/235) and now called αSpIIΣI/βSpIIΣ1, was found to be primarily located in the axons and presynaptic terminals of all neurons.^68–70 Brain spectrin isoforms are (αβ)₂ tetramers.^71,72 αSpI/βSpIΣ2 had binding sites for erythroid ankyrin, protein 4.1, and actin, which are colocalized in the neuronal soma, dendrites, and postsynaptic densities.^64–66,73 In the case of αSpIIΣI/βSpIIΣ1, the tetramer had binding sites for nonerythroid isoforms of protein 4.1 and ankyrin, but also had association sites for calmodulin and synapsin.^{64–66,74,75}

An immunoelectron microscopy study from Zagon et al.⁷⁶ led to the interesting conclusion that the spectrin isoforms were located on the cytoplasmic surface of not only the plasma membrane but also all organelle membranes, and within the nucleus. They were also cross-linking cytoskeletal structures to each other and membrane surfaces. The location of spectrin isoforms in brain is summarized in Figure 4. Of great interest in this immunoelectron microscopy study was the finding of αSpIIΣI/βSpIIΣ1 associating with the cytoplasmic surface of small spherical synaptic vesicles. Lambert and colleagues have demonstrated that αSpII can be found within the nucleus where it plays an important role in DNA repair.^77–81

Table 2.

Spectrin isoform nomenclature and properties.

Spectrin isoform	Previous names	Molecular weight	mRNA size	Genomic locus	Chromosome
					Mouse	Human
αSpIΣ1	α Erythrocyte spectrin	280 kDa	8 kb	Spna1	1	1
αSpIIΣ1	Nonerythroid	280 kDa	7.8 kb	Spna2	2	9
Σ2	α Spectrin
Σ3	α Fodrin
Σ4
βSpIΣ1	β Erythrocyte	246 kDa	6/8 kb	Spnb1	12	14
βSpIΣ2	β Erythrocyte spectrin	268 kDa	11 kb	Spnb1	12	14
βSpIIΣ1	Nonerythroid	275 kDa	9 kb	Spnb2	11	2
Σ2	β Spectrin
	β fodrin
βSpIIIΣ1	–	271 kDa	8–9 kb	Spnb3	19	11
βSpIVΣ1	–	288 kDa	9 kb	Spnb4	7	19
Σ2
Σ3
Σ4
βSpVΣ1	–	417 kDa	11–12 kb	Spnb5	–	15

Figure 4.

Location of spectrin isoforms. Red indicates the position of spectrin αI/βIΣ2 and blue αII/βII in neurons. Taken from Zagon et al.⁷⁶ with permission of the publisher

It has been demonstrated that the connection between spectrin and the small spherical synaptic vesicles was end-on via synapsin.^74,82,83 The Goodman lab also demonstrated that the brain spectrin–synaptic vesicle interaction is not regulated by phosphorylation of synapsin by Cam Kinase II or A-Kinase,⁸⁴ but is directly regulated by free Ca²⁺.⁶⁶ Ma et al.⁸⁵ cloned and sequenced βSpIIΣ1 spectrin from mouse, which demonstrated that βSpIIΣ1 had 2363 residues that had 59% identity with βSpIΣ1. This sequence identity rose to 89% in the actin-binding domain, and to 87% in residues 207–445, which was predicted to be the synapsin-binding domain. The suggestion by Ma et al.⁸⁵ proved to be correct based on microinjection of peptide-specific antibodies against this region of βSpIIΣ1 into paired hippocampal neurons.⁸⁶ Importantly, Sikorski et al.⁸⁶ demonstrated that when these antibodies were injected into the presynaptic neuron, this inhibited excitatory postsynaptic currents (EPSCs) in the postsynaptic neuron. Peptide-specific antibodies against flanking sequences had no effect upon EPSCs. The attachment site for synapsin on βSpII was mapped to L211–Q235 by Zimmer et al.⁸⁷ Based on these, and other studies, we concluded that αSpII/βSpII serves an essential role as a docking protein for Ca2+-regulated exocytosis of neurotransmitter at the active zone of the nerve terminal.⁶⁶

Potential regulatory role for spectrin in nonerythroid cells

While αSpIIΣI/βSpIIΣ1 spectrin is found in all cell types except RBCs, αSpI/βSpIΣ1 or 2 are found only in RBCs, neurons, skeletal muscle, and cardiac muscle (Table 2).^{54,66,68,70,88–91} In human, there are two α spectrin genes: SPTA1, SPTAN1-encoding αSpI and αSpIIΣI, respectively, and five genes encoding β spectrins: SPTB, SPTBN1, SPTBN2, SPTBN4, and SPTBN5. We have been discussing the gene products of SPTB, SPTBN1 (βSpIΣ2 and βSpIIΣ1, respectively), which are the major isoforms found in brain. Clark et al.⁷⁰ demonstrated that hybrid tetramers of spectrin subunits also exist in the brain. These hybrid species are less abundant than tetramers consisting solely of erythroid subunits (αSpI/βSpIΣ2)₂ or nonerythroid subunits (αSpIIΣ1/βSpIIΣ1)₂. The hybrid tetramers provide an explanation of how two α spectrin isoforms (αSpI and αSpII) can couple with five different β spectrin isoforms (βSpI-V).

We anticipate that the αSpI subunit found in neurons, skeletal, and cardiac muscle^{54,66,68,70,88–91} would have the same chimeric E2/E3 activity as this subunit found in RBCs, but with a broader range of targets. While logical this has not yet been tested. In RBCs, spectrin’s targets include ankyrin, protein 4.1, protein 4.2, adducin, the anion transport channel, and protein gi13278939. Therefore, it would be pertinent to examine whether spectrin possesses ubiquitination activity with these proteins or their analogs in cardiac and skeletal muscle cells as well. αSpI and αSpII expressed in cardiac muscle cells are found on the plasma membrane and within contractile fibers near the Z-disc and intercalated disc.^89,92 In mice, it has also been shown that protein 4.1 products (4.1R,G,N,B) occur with spectrins at many subcellular locations in the heart, along with ankyrins (AnkB, AnkR, AnkG) and actins.⁹³ The significance of the spectrin membrane cytoskeleton and the ubiquitin-proteasome system in maintenance of myocyte integrity is illustrated by linkage of cardiac pathologies with mutations in these cellular components.^94,95 Thus, examining spectrin chimeric ubiquitin ligase activity in heart cells will promote a better understanding of its contribution to cardiac protein turnover, protein–protein interactions, and cellular signaling in cardiac pathophysiology.

In the case of αSpIIΣI, we know that it shares sequence homology with αSpI in the region surrounding a cysteine equivalent to Cys 2071 (Figure 5). We also know that both αSpI and αSpIIΣI are ubiquitinated in the hippocampal neurons.^96–98 αSpIIΣI shares sequence homology with αSpI in the region surrounding a cysteine equivalent to Cys 2071 (Figure 5). Moreover, there is an absolute conservation of the Cys 2071 sequence domain in eukaryotes throughout evolution, highly suggestive of conservation of a functional domain within the αSpII molecule. There have been no reported studies on whether αSpIIΣI can serve as an E2/E3 chimeric enzyme in nonerythroid cell types. This is an extremely important question as spectrin makes up ∼2–3% of the total protein in nonerythroid cells. We would predict that it will have such chimeric E2/E3 enzymatic activity, with far greater number of target proteins, for several reasons: (1) It is found on the cytoplasmic surface of both organelle and plasma membranes,^76,99 and within the nucleus.^77–81 (2) αSpII is directly associated with many cytoskeletal and membrane skeletal components,^{2,5,6,63–67,100–102} and cell adhesion proteins (e.g. NCAM180).^{2,5,6,63–67,103} (3) It has indirect interaction, via protein 4.1 and ankyrin, to a myriad of ion channels and transporters.^{2,5,6,44,63–67,104,105} (4) αSpII is expressed throughout all mammalian developmental stages.^106–108 (5) The RBC proteomic and interactomic analyses to date have indicated 2289 unique proteins, with αSpI/βSpI having moderate connectivity.^109–112 The RBC has no internal organelles except 20 S proteasomes.¹¹³ The proteome of nucleated nonerythroid cells, with a complete complement of organelles, will have at least 10 times the number of unique proteins, as compared with RBCs, with far greater connectivity of αSpII/βSpII spectrin. Further study of the E2/E3 activity of α spectrin isoforms in nonerythroid cells is of great importance to our understanding the ubiquitination of many proteins and the cellular functional impact of this posttranslational modification.

Figure 5.

E2/E3 chimeric active sites. Conservation of sequence around the active site 2071Cys of human αSPI. In (a), the sequence surrounding 2071Cys is shown in the upper line with the amino acids conforming to E2 ubiquitin-conjugating enzyme active sites are underlined. The region of the nonerythroid αSpII that demonstrates a ∼63% identity to αSpI is denoted in the lower line with the sequence identical to αSpI shown in red. Cys2071 is shown by the arrow. In (b), the region of human aSpII containing the putative E2 site was used to search databases using tBlastn and Tfasa programs. This analysis demonstrated a very high identity of the region conserved in sequence and location within the various spectrin molecules. Illustrated here are the sequences for mouse (P16546), chicken (X02593.1), Drosophila (XM_002026155.1), and Caenorhabditis elegans (LK928133.1). All are identical to the human aSpII except for Drosophila with an isoleucine substituted for a leucine. The arrow denotes the potential active site Cys for these proteins

Authors’ contributions

All authors participated in the writing and editing of the manuscript. SRG and WEZ serve as co-corresponding authors on the final manuscript.

Conflict of interest

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.