Download MNS Blood Group System variants on Malarial Resistance

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, 4q28à4q31)
•  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, 4q28à4q31)
•  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
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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
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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