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MNS Blood Group System Biochemistry, genetics, evolution, and the role of glycophorins and populational variants on Malarial Resistance Jesse Qiao, M.D. Pathology Seminar Series 1/28/13 Introduction to the MNS System • Second blood group system discovered • Second in complexity compared w/ Rh • M and N is one allele (gene pair) Phenotypes in Caucasians: M+ N- : 28% M+ N + : 50% M- N+ : 22% • S and s is one allele (gene pair) Phenotypes in Caucasians: S+ s- : 11% S+ s+ : 44% S- s+ : 45% • Prevalence of phenotypes is different in African Americans (& other ethnicities) MN and Ss alleles – wild type Basic Sciences Refresher Pseudo-Genetics 3301* Course Pseudo-Biochemistry 3301* Course *3XXX series: courses taken at the junior level in college Central Dogma of Molecular Biology GENES “IMMATURE” PROTEINS “MATURE” PROTEINS Post-‐Transla8onal Modifica8on (e.g. glycosyla8on) Basic Concepts • Exon: DNA that encodes protein parts • Intron: DNA that does NOT encode protein parts • Gene: A segment of DNA containing exons & introns. - Introns are removed (“spliced”) - Exons are joined, forming a mRNA for translation • Allele: Variations of a gene (e.g. C/c E/e) • 5’ : start of a DNA sequence • 3’ : end of a DNA sequence • Mutations: any change in DNA sequence Insertion: adds new DNA from sequence Deletion: truncates DNA from sequence Point: change of DNA sequence w/o affecting qty Conversion vs. Recombination • Gene Conversion: Segment of genetic material from one chromosome is copied onto the other without changes in the donor chromosome. • Recombination (crossover): Breaking and re-joining of both DNA strands to form new molecules of DNA with both chromosomes with different genetic information. Basic Concepts • Protein: complex arrangement of a linear sequence of amino acids • Peptide: another term for “protein” • N-terminus: start of a protein sequence • C-terminus: end of a protein sequence • Post-translational modification: the newly translated protein undergoes further processing • Cell membrane: a lipid bilayer that is hydrophobic (“hates water”) Amino Acids Building Blocks of Proteins COLOR CODE: Beige: non-polar (“hates water”) Green: polar, non-charged Purple: acidic, polar, negatively charged Blue: basic, polar, positively charged Protein Structure • Primary structure: Amino Acid Sequence • Secondary structure: Sub-structure (helix or sheets) Forming domains • Tertiary structure: 3D shape of protein unit (e.g. GYP A, RHD) • Quaternary structure: groups of protein units (e.g. RHD-RHAG heterodimers) • All structural categories are related & play a role in antigen formation & subsequent development of antibodies!! Protein Glycosylation • New, translated protein • Attachment of a complex sugar molecule onto the protein N- link to nitrogen molecule O- link to oxygen molecule • Can alter / change the tertiary and/or quaternary structure of proteins • Glycosylation affects antigenicity of translated proteins regardless of changes in the primary sequence!! Glycophorins Wild Type, Normal Structure, and Biochemical Structure Glycophorin Structure • C-terminal region (cytoplasmic) - anchor to other intra-cellular proteins • N-terminal region (extracellular) - amino acid sequence(s) responsible for antigen specificity (M, N, ‘N’, S, s) - Ser (S) and Thr (T) with O-linked glycans that contain sialic acid residues attached to galactose • Transmembrane region - contains mostly non-polar amino acids. Glycophorin A: - Expresses M or N antigen (amino acids 1-5) EC Domain Transmembrane Domain Cytoplasmic Domain Glycophorin B: - Expresses S or s antigen (amino acid 29) - Expresses ‘N’ (amino acids 1-5) ‘N’ is NOT a true antigen!!!! tertiary structure prevents antigen from being detected… YET, because one has the amino acid sequence, the body does not recognize N as foreign when transfused with N+ red cells. Topographic Arrangement of GYP genes (Chromosome 4, 4q28à4q31) • 5’ : • In between: • 3’ : GYPA GYPB GYPE Glycophorin A, B, E Exon Configuration GYP A / B Exons & Corresponding Amino Acid Sequences Glycophorin Exons Glycophorin A: - Expresses M or N antigen (amino acids 1-5) EC Domain Transmembrane Domain Cytoplasmic Domain Glycophorin B: - Expresses S or s antigen (amino acid 29) - Expresses ‘N’ (amino acids 1-5) ‘N’ is NOT a true antigen!!!! tertiary structure prevents antigen from being detected… YET, because one has the amino acid sequence, the body does not recognize N as foreign when transfused with N+ red cells. Glycophorin A Amino Acid Sequence Glycophorin B Amino Acid Sequence Sialic Acid, Galactose, and O-linked glycans • Sialic acid (aliases: N-acetylneuramic acid, Neu5Ac) - abundant on glycophorins & other glycoproteins - antigenic determinants on GPA and B - Linkage on the O-glycan: Neu5Ac—Galactose via (α2,3)-, (α2,6) (next slide) Figure 3. Sialic acid. The glycosidic linkage forms at carbon #2, since it is part of a hemiacetal. This reacts with the OH group of another sugar molecule. Because the #3 and #6 carbons of sialic acid have no OH groups and thus do not lend themselves to glycosidic bond formation, sialic acid normally attaches only as a terminal residue in the saccharide chains (the OH at carbon 8 does, however, sometimes participate in glycosidic linkages). Its terminal positioning gives it a crucial role in immune system recognition and cell adhesion Source: http://www.crscientific.com/article-5-min-stain-CrO3.html Linkage of Sialic Acid (Neu5Ac) to Galactose Formation of Low Frequency Antigens An Overview Genetic alterations within MNS 1. Gene deletion - either Glycophorin A and/or B are not expressed 2. Unequal crossing over (recombination) - between homologous but NOT identical genes - resembles the GYP evolutionary mechanism 3. Gene conversion - point mutations, gene replacement, or altered repair after DNA damage - affects the antigenic sequences - formation of low frequency antigens (LFAs) Remember that ‘N’ is ‘never’ a true antigen!!! Topographic Arrangement of GYP genes (Chromosome 4, 4q28à4q31) • 5’ : • In between: • 3’ : GYP A GYP B GYP E 1. GYP Gene Deletions • En-: deletion à no functional Glycophorin A - Loss of phenotypes: M+ or N+ • U-: deletion à no functional Glycophorin B - Loss of phenotype: ‘N’+ S+ or s+ • Mk: deletion à no Glycophorin A and B - Phenotype: null (GYPE expressed, significance unknown) 2. Unequal Cross-Over of GYP A & B 3. Gene Conversion à Hybrid GPs [ GYP (A-B-A) and GYP (B-A-B) genes] GYP (A-B-A) Hybrid Gene - Example Formation of He (Henshaw) antigen • 3% of African Americans • Two-step process: 1. Replacement of distal exon B2 on GYPB by: - homologous segment of distal exon A2 - proximal intron after A2 2. Subsequent random mutations in the replaced segment • Result: loss of normal ‘N’ on Glycophorin B GYP (A-B-A) Hybrid Gene - Example Formation of He (Henshaw) antigen Low Frequency Antigens Associated w/ Abnormal Glycosylation • Africans > Caucasians • Specificity of anti-M and anti-N are partially dependent on the attached sugar residues (O-glycans and Nglycans) • Examples of such LFA’s: Hu, Sext, M1, Tm, Sj, Can - Attaching O-glycan to GPA/B peptides requires help from glycosyltransferases - Mutations in genes producing glycosyltransferases affect O-glycan attachment, which then affects antigenicity • Neu5Ac-(α 2,6)-Gal-…-- (Sialic acid O-glycan attached to residues 2-4 of GPA and GPB) are substituted by Nacetylglucosamine-- on some LFAs of GPA Neu5Ac (sialic acid)-‐glycan-‐-‐ Ser/Thr replaced by N-‐acetylglucosamine-‐-‐ Evolution and Function of the Glycophorins Evolution of Glycophorin Genes How did we get 3 genes, A, B, & E? • Ancestry: ONE gene, Glycophorin A (GYP A) • Step 1: Duplication of a segment of GYP A • Step 2: Duplication of everything above • Step 3: Unequal X-over à “GYP B/E” • Step 4: Duplication of “GYP B/E” • Step 5: Mutation into GYP B and E Development and Function of GPA/B • GPA/B Normally expressed on red cells only • Abundant sialic acid content (negative charge) - prevent spontaneous RBC aggregation (“like repel”) • Heavily glycosylated… hence glycophorin • Contribute to glycocalyx (“cell coat”) - protects cell from damage and microbial attack • Associated with anion transporter on the membrane • Signal transduction & interaction w/ membrane skeleton • Limited complement regulator: inhibiting binding of C5b-7 • GPA/B is not “required” for good health What about Glycophorins C and D? • Glycophorin A, B, E: MNS system (Chromosome 4) • Glycophorin C, D: Gerbich system (Chromosome 2) • Thus, GYPC has NO homology with GYPA, B, E - Alternate splicing: variants in phenotype - Truncated translation within GYPC mRNA: Glycophorin D (truncated GPC, - 1st 21 AAs) - Point mutations within GYPC: LFAs - Gerbich, Webb, Dutch, Ahonen http://www.uniprot.org/blast/?about=P04921[1-21] Malaria and Glycophorins Malaria - Overview • • • • • • Phylum: Protozoa Class: Sporozoa Order: Coccidiida Family: Plasmodiidae Genus: Plasmodia Species affecting humans: - P. vivax - P. ovale - P. falciparum - P. malariae - P. knowlesi – SE Asia, more simian than humans Krief S et al. On the Diversity of Malaria Parasites in African Apes and the Origin of P. falciparum from Bonobos. PLoS Pathog 2010;6(2): e1000765. Domain, Kingdom, Phylum, Class, Order, Family, Genus, Species SOURCE: WIKIPEDIA • To remember the order of taxa in biology: ▫ Dumb Kids Prefer Candy Over Fancy Green Salad ▫ Dumb Kids Playing Catch On Freeway Get Squashed. ▫ "Dear King Philip Come Over For Good Spaghetti/ Soup" is often cited as a (clean) method for teaching students to memorize the taxonomic classification system.[2] Other variations tend to start with the mythical king, with one author noting "The nonsense about King Philip, or some ribald version of it, has been memorized by generations of biology students." [3] ▫ Dumb “King Plays Chess On Fat Girl's Stomach" http://www.malariasite.com/malaria/MalarialParasite.htm Plasmodia Species RBC antigens & Ports of Entry • Duffy – P. vivax – ligand & chemokine-mediated entry - well researched • ABO – P. falciparum – A and B are stronger rosetting receptors than O • Knops – antigens are prorosetting • MNS – P. falciparum Glycophorin A, B • Gerbich - P. falciparum Glycophorin C • Rh – V antigen • KEL – Js-a Rowe et al. Plasmodium Falciparum the most deadly of all… Rowe et al. Figure 1. Life cycle of Plasmodium falciparum When an infected female Anopheles mosquito takes a blood meal, sporozoite forms of Plasmodium falciparum are injected into the human skin. The sporozoites migrate into the bloodstream and then invade liver cells. The parasite grows and divides within liver cells for 8–10 days, then daughter cells, called merozoites, are released from the liver into the bloodstream, where they rapidly invade red blood cells (RBCs). Merozoites subsequently develop into ring, pigmented-trophozoite, and schizont stage parasites within the infected RBC. P. falciparum-infected erythrocytes express parasite-derived adhesion molecules on their surface, resulting in sequestration of pigmented-trophozoite and schizont stage-infected RBCs in the microvasculature. The asexual intraerythrocytic cycle lasts 48 h and is completed by the formation and release of new merozoites that will reinvade uninfected RBCs. It is during this asexual bloodstream cycle that the clinical symptoms of malaria (fever, chills, impaired consciousness, etc.) occur. During the asexual cycle, some of the infected RBCs develop into male and female sexual stages called gametocytes that are available to be taken up by feeding female mosquitoes. The gametocytes are fertilized and undergo further development in the mosquito, resulting in the presence of sporozoites in the mosquito’s salivary glands ready to infect another human host. Reproduced with permission from [1]. P. vivax vs. P. falciparum • P. vivax has only one port of entry into RBCs - Duffy antigen on the host RBCs - Duffy Binding Protein (DBP) on P. vivax - Duffy A & B neg persons: resistant to P. vivax • P. falciparum has highly redundant, alternate invasion pathways using different receptors - Deficiency in one of the glycophorins does NOT lead to complete resistance to infection Ports of Entry P. vivax vs P. falciparum Organism Ligand (on parasite) Receptor (on RBC) P. vivax DBP Duffy A, Duffy B P. falciparum EBA-175 Glycophorin A (M, N) P. falciparum EBA-140 Glycophorin C (Gerbich) P. falciparum EBA-181 not known P. falciparum EBA-1 Glycophorin B (S, s, U+) DBP = Duffy Binding Protein EBA = Erythrocyte Binding Antigen EBA-175 and GPA Reference Article: Orlandi et al (1992) • Binding of EBA-175 requires N-acetylneuramic acid (Sialic acid, Neu5Ac) linked to O-glycan on GPA - Neu5Ac(α2-3)-Gal-…-O-Ser or Thr on N-term • Cleavage of the O-linked oligosaccharides on the N-terminus of GPA à marked decrease in EBA-175 binding activity EBL-1 and GPB Reference Article: Mayer et al. (2008) • No specific P. falciparum ligand has been previously associated with GPB • However, increased GPB polymorphisms (higher incidence of LFAs) in malaria endemic regions in Africa - loss of GPB (U-), altered AAs (e.g. Henshaw) • Although no specific mechanisms described, through enzymatic digestion studies EBL-1 is specific for GPB (will not bind GPA) Conclusion • Understanding the genetics of the MNS system is crucial to explaining the molecular variations • Numerous MNS molecular variations exist (beyond the topic of today’s discussion) • Clinical significance of many variants with respect to transfusion medicine remains undiscovered • Mechanisms for malarial invasion and resistance to infection still under investigation Studies on the MNS focused on the Caucasian and African population. What about Asians/Latinos? Some Latinos are of mixed ancestry - Asian (Native American) & European The Bering Strait (about 50 mi/80 km) Once land, now ice/water It’s summer time… Would you want to drive on this thing? References Primary Reference Text: • Daniels, Geoff. Human Blood Groups, Second Edition. Blackwell Publishing Company, 2002. Additional Text References • A Malaria Invasion Receptor, the 175-Kilodalton Erythrocyte Binding Antigen of Plasmodium falciparum Recognizes the Terminal Neu5Ac(a2-3)GalSequences of Glycophorin A Palmer A. Orlandi, FrancisW. Klotz, and J. David Haynes. Feb 15 1992. • Mayer, G. et al. Glycophorin B is the erythrocyte receptor of plasmodium falciparum erythrocyte-binding leigand, EBL-1. PNAS, 3/31/2009. Vo 106 #13 Primary Source of Images: • Daniels, Geoff. “MNS System”, Chapter 3, Human Blood Groups, Second Edition, pp. 99-174. Blackwell Publishing Company, 2002. The images from the text above are in black and white and has been scanned. Additional color has been added for organization and consistency: M: dark blue N: standard blue ‘N’: red S: dark green s: teal cytoplasmic domain of GPA/B: purple transmembrane domain of GPA/B: gray extracelluar domain of GPA/B: green Additional Source(s) of Images: • General search on Google Images on the relevant topics of discussion • Wikipedia • Henry’s Clinical Diagnosis & Management by Laboratory Methods, 21st Ed, B Saunders, 2006. • http://njms2.umdnj.edu/biochweb/education/bioweb/PreK2010/AminoAcids.htm • J. Alexandra Rowe, et al. Blood groups and malaria: fresh insights into pathogenesis and identification of targets for intervention. Curr Opin Hematol. 2009 November ; 16(6): 480–487. • http://www.malariasite.com/malaria/MalarialParasite.htm • Varki, A. Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins. Nature, Vol 446 , 26 Apr 2007 • http://effectmeasure.blogspot.com/2006/01/background-science-for-turkish_18.html