membranes Review A Comprehensive Review of Our Current Understanding of Red Blood Cell (RBC) Glycoproteins Takahiko Aoki Laboratory of Quality in Marine Products, Graduate School of Bioresources, Mie University, 1577 Kurima Machiya-cho, Mie, Tsu 514-8507, Japan; [email protected]; Tel.: +81-59-231-9569; Fax: +81-59-231-9557 Received: 18 August 2017; Accepted: 24 September 2017; Published: 29 September 2017 Abstract: Human red blood cells (RBC), which are the cells most commonly used in the study of biological membranes, have some glycoproteins in their cell membrane. These membrane proteins are band 3 and glycophorins A–D, and some substoichiometric glycoproteins (e.g., CD44, CD47, Lu, Kell, Duffy). The oligosaccharide that band 3 contains has one N-linked oligosaccharide, and glycophorins possess mostly O-linked oligosaccharides. The end of the O-linked oligosaccharide is linked to sialic acid. In humans, this sialic acid is N-acetylneuraminic acid (NeuAc). Another sialic acid, N-glycolylneuraminic acid (NeuGc) is present in red blood cells of non-human origin. While the biological function of band 3 is well known as an anion exchanger, it has been suggested that the oligosaccharide of band 3 does not affect the anion transport function. Although band 3 has been studied in detail, the physiological functions of glycophorins remain unclear. This review mainly describes the sialo-oligosaccharide structures of band 3 and glycophorins, followed by a discussion of the physiological functions that have been reported in the literature to date. Moreover, other glycoproteins in red blood cell membranes of non-human origin are described, and the physiological function of glycophorin in carp red blood cell membranes is discussed with respect to its bacteriostatic activity. Keywords: red blood cell; glycoproteins; biological function; glycophorin; band 3; O-linked oligosaccharides; N-acetylneuraminic acid; N-glycolylneuraminic acid 1. Introduction The blood of mammals, such as humans, as well as of birds, reptiles, and teleosts, contains red blood cells (erythrocytes). Human red blood cells, which are the most commonly studied by researchers, are approximately 7 µm in diameter, and their centre has a dented discoid shape. Studies on biological membranes normally use human red blood cells (RBC) because these erythrocytes have no nuclei and are easy to obtain for cell membrane preparation. For the preparation of membrane proteins, it is necessary to solubilize the phospholipid bilayer by using detergents. Fairbanks et al. [1] developed a method in which red blood cell membranes were solubilized by sodium dodecyl sulfate (SDS), and then the extracted membrane proteins were separated by acrylamide gel electrophoresis (SDS-PAGE). As a result, membrane proteins and glycoproteins could be detected on SDS gels. Figure1 shows the typical human red blood cell membrane proteins separated by SDS-PAGE using the method of Laemmli [2], which was later improved by Fairbanks et al. [1]. Table1 depicts the nomenclature of each cell membrane protein according to Fairbanks et al. [1], along with apparent molecular mass (kDa) and physiological function. Band 3, band 4.1, and band 4.2 are called the designated names at present. Membranes 2017, 7, 56; doi:10.3390/membranes7040056 www.mdpi.com/journal/membranes Membranes 2017, 7, 56 2 of 19 Membranes 2017, 7, 56 2 of 18 FigureFigure 1. 1. SodiumSodium dodecyl dodecyl sulfate sulfate (SDS) (SDS)-polyacrylamide-polyacrylamide gel gelelectrophoresis electrophoresis of human of human red blood red blood cell (RBC)cell (RBC) membranes. membranes. Healthy Healthy human human RBC RBC membranes membranes stained stained with with Coomassie Coomassie brilliant brilliant blue. blue. ElectrophoresisElectrophoresis was performed according to the method of Laemmli [[2].2]. TableTable 1. Red blood cell membrane pro proteins.teins. Band Molecular Mass (kDa) Designation Function 1 Band240 Molecular Mass (kDa)spectrin (α Designation chain) Function 2 1220 240spectrin spectrin (β chain) (α chain) components of cytoskeleton components of cytoskeleton 2.1 2210 220ankylin spectrin (β chain) 3 2.1100 210band 3 (AE1) ankylin anion transporter 4.1 382 100band band 4.1 3 (AE1) anion transporter components of cytoskeleton 4.1 82 band 4.1 4.2 76 band 4.2 components of cytoskeleton 4.5 4.255 76band 4.5 (GLUT1) band 4.2 glucose transporter 5 4.543 55actin band 4.5 (GLUT1)components glucose transporter of cytoskeleton 6 535 43glyceraldehyde-3-phoshate dehydrogenase actin (GAPDH) componentsglycolytic of cytoskeleton enzyme 7 631 35 glyceraldehyde-3-phoshatestomatin dehydrogenase (GAPDH) glycolytic– enzyme 7 31 stomatin – Band 3 is a glycoprotein and is detected as a diffuse band on SDS gel due to the microheterogeneityBand 3 is a glycoprotein of the attached and is detectedoligosaccharides as a diffuse [3]. band Band on 3 SDSis detectable gel due to on the the microheterogeneity SDS gel by using proteinof the attached staining oligosaccharides with Coomassie [3]. Brilliant Band 3 is Blue detectable (CBB) on because the SDS band gel by 3 using has proteina small staining amount with of oligosaccharides.Coomassie Brilliant Band Blue 3 (CBB)contains because approximately band 3 has 7% a small carbohydrate amount of and oligosaccharides. contributes toBand approximately 3 contains 10%approximately of the total membrane 7% carbohydrate carbohydrate and contributes [4]. By contrast, to approximately the sialooligosaccharide 10% of the-rich total glycoproteins membrane arecarbohydrate detectable [4by]. Bystaining contrast, with the the sialooligosaccharide-rich periodic acid-Schiff’s glycoproteins(PAS) reagent are[1,5]. detectable The glycoproteins by staining detectedwith the by periodic PAS staining acid-Schiff’s are called (PAS) glycophorins. reagent [1,5]. Figure The glycoproteins 2 shows the detectednomenclature by PAS of staininghuman red are cellcalled membrane glycophorins. sialoglycoproteins. Figure2 shows theThe nomenclature designation of of humanglycophorins red cell is membrane confusing sialoglycoproteins.because different nomenclaturesThe designation have of glycophorinsbeen used by different is confusing researchers. because In different the first nomenclaturespart of the 1970s, have the beenglycophorins used by weredifferent called researchers. as PAS 1 In–4 theand, first at partpresent, of the they 1970s, are thetermed glycophorins glycophorins were A called–D, respectively as PAS 1–4 and,[6]. Glycophorinat present, they α is the are same termed as glycophorin glycophorins A, A–D, and glycophorin respectively δ, [ 6β,]. and Glycophorin γ are glycophorinsα is the B, same C, and as D,glycophorin respectively A, [6 and–8]. glycophorin δ, β, and γ are glycophorins B, C, and D, respectively [6–8]. GlycophorinGlycophorin A A (dimer) (dimer) is is o observedbserved below below band band 3 3 on SDS SDS-polyacrylamide-polyacrylamide gels gels (Figure (Figure 22),), isis aa majormajor component component of of red red cell cell membrane membrane glycoproteins, glycoproteins, and and is is reported reported to to have have an an apparent apparent molecular molecular massmass of 29 [9] [9] to 36 kDa [10,11]. [10,11]. TheThe electrophoreticelectrophoretic migration migration of glycophorinof glycophorin is relatively is relatively low when low compared when compared to other membrane to other membranecomponents components because glycophorin because glycophorin is heavily glycosylated. is heavily glycosylated. Although the Although molecular the mass molecular of the mass other ofmembrane the other proteins membrane can estimatedproteins can by migrationestimated on by SDS-PAGE, migration theon SDS molecular-PAGE, mass the ofmolecular each glycophorin mass of eachcannot glycophorin be estimated cannot in this be manner.estimated in this manner. Membranes 2017, 7, 56 3 of 19 Membranes 2017, 7, 56 3 of 18 Figure 2. SDS-polyacrylamide gel electrophoresis of human red blood cell (RBC) membranes. Healthy Figure 2. SDS-polyacrylamide gel electrophoresis of human red blood cell (RBC) membranes. Healthy human RBC membranes stained with periodic acid-Schiff (PAS) stain [1]. Electrophoresis was human RBC membranes stained with periodic acid-Schiff (PAS) stain [1]. Electrophoresis was performed according to the method of LaemmliLaemmli [[2].2]. 2. Structure of the Human Red Blood Cell Membrane 2. Structure of the Human Red Blood Cell Membrane Red blood cell membrane proteins [12–14] can be classified into transmembrane proteins and Red blood cell membrane proteins [12–14] can be classified into transmembrane proteins and peripheral membrane proteins that associate with the cytoplasmic side of the lipid bilayer. Band 3 peripheral membrane proteins that associate with the cytoplasmic side of the lipid bilayer. Band 3 and and glycophorins are transmembrane proteins. Transmembrane proteins associate with the glycophorins are transmembrane proteins. Transmembrane proteins associate with the peripheral peripheral proteins that constitute the protein meshwork (cytoskeleton). Figure 3 shows a schematic proteins that constitute the protein meshwork (cytoskeleton). Figure3 shows a schematic drawing drawing of the site of band 3 and glycophorins in human red blood cell membrane structure, with of the site of band 3 and glycophorins in human red blood cell membrane structure, with reference reference to the review on the red cell membrane skeleton [14,15]. The main component of the to the review on the red cell membrane skeleton [14,15]. The main component