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HEMOGLOBIN M- SASKATOON: A Variant with Reduced Oxygen
Binding Abilities and Cyanosis
Patrick Reed and Janet Murray, UVM, Fall 2008
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Hemoglobin
Hemoglobin (Hb) is the protein in red blood cells that
reversibly binds oxygen and carries it from the lungs to
all the cells of our body. It is the essential oxygen
delivery protein in all animals. HbA, the form found in
adult humans, is a tetrapeptide protein composed of two
alpha chains (gene located on chromosome 16) and two
beta chains (gene located on chromosome 11). Each
peptide chain has one heme molecule bound to it. Each
heme molecule has one atom of iron in the center of a
protoporphyrin ring. The iron binds oxygen reversibly.
To do so, iron must be in its reduced valence state
(Fe+2). Methemoglobin (metHb) is the term used to
describe hemoglobin in which the iron is in the oxidized
valence state (Fe+3) and is unable to bind with oxygen.
Clinical Information:
Hemoglobin M-Saskatoon
Hemoglobin M-Saskatoon is a beta peptide mutation and is one of
seven known variants of hemoglobin in which the patient exhibits
cyanosis (blue skin color) due to the presence of high levels of
methemoglobin (metHb) in the red blood cells. For this reason the
hemoglobin variants found in these patients are referred as “M”
hemoglobins.
Hb M-Saskatoon was first described in a Canadian family (in
Saskatoon, CA) in 1956. Subsequently additional reports in 1978
and 1985 from Italy and India showed the same condition, implying
the genetic change had occurred independently several times
during human evolution.
Hemoglobin M-Saskatoon has one allele where Histidine 63 is
replaced by a Tyrosine (His63Tyr or H63Y). This leads to an
accumulation of MetHb of up to 30 to 50%. MetHb has a
blue/chocolate color which makes the patients skin look blue,
called cyanosis. Inheritance patterns for Hb M variants are
consistently heterozygous suggesting that the homozygous form
is incompatible with life.
1 mvhltpeeks avtalwgkvn vdevggealg rllvvypwtq rffesfgdls tpdavmgnpk
61 vkahgkkvlg afsdglahld nlkgtfatls elhcdklhvd penfrllgnv lvcvlahhfg
121 keftppvqaa yqkvvagvan alahkyh
63rd amino acid--histidine
Beta hemoglobin is a 147 amino acid protein:
note that the final molecule is only 146 amino
acids long. The first methionine is removed posttranslationally. The 63rd amino acid in the final
protein, histidine (H), is highlighted in red. In Hb
M-Saskatoon, this amino acid is a tyrosine (Y).
A
Organism KEY
HBB_PANTR _ Chimpanzee
HBB_PANPA _ Pygmy chimpanzee (Bonobo)
HBB_HUMAN _ Human
HBB_GORGO _ Lowland gorilla
HBB_HYLLA _Common gibbon
HBB_SEMEN _ Hanuman langur
HBB_ATEPA _ [Black spider monkey
HBB_PITPI _ White-faced saki
HBB_CEBAP _ Brown-capped capuchin
HBB_RABIT _ Rabbit
HBB_BALAC_Minke whale
HBB_MACGG _ Australian ghost bat
HBB_PIG _ Pig
Conserved histidine at position
63 in final protein
B
The human beta hemoglobin gene
C
LOCUS
NM_000518
626 bp mRNA linear PRI 12-OCT-2008
DEFINITION Homo sapiens hemoglobin, beta (HBB), mRNA.
ACCESSION NM_000518
VERSION NM_000518.4 GI:28302128
chromosome: 11; Location: 11p15.5
The human beta hemoglobin gene (HBB) consists of three exons
and two introns and is 1606 bases.
Exon 1, 1-142 (142 bp)
Intron 1, 143-272 (130 bp)
Exon 2 273-495 (223 bp)
Intron 2, 496-1345 (850)
Exon3 1346-1606 (261 bp)
The mRNA is 626 bases long. Coding sequence (CDS) is from
base 51 to base 494.
Exon 1, 1-142 (includes 50 base 5’ UTR) (142 bases)
Exon 2, 143-365 (223 bases)
Exon3, 366-626 (Includes 132 base 3’ UTR) (261 bases)
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removed from final protein
Cyanosis due to the presence
of up to 50% MetHb. Note blue
skin hues especially on lips
and neck.
Heme molecule showing the iron atom at the center with
bonding coordinates with the 4 nitrogen atoms of the
protoporphyrin ring The iron atom is held within the
heme molecule by two histidine amino acids of the
peptide chain.
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Amino Acid Sequence and Conservation of the Human
Beta Hemoglobin Protein
NCBI data for a 626 bp mRNA transcript
ORIGIN
1 acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc atggtgcatc
61 tgactcctga ggagaagtct gccgttactg ccctgtgggg caaggtgaac gtggatgaag
121 ttggtggtga ggccctgggc aggctgctgg tggtctaccc ttggacccag aggttctttg
181 agtcctttgg ggatctgtcc actcctgatg ctgttatggg caaccctaag gtgaaggctc
241 atggcaagaa agtgctcggt gcctttagtg atggcctggc tcacctggac aacctcaagg
301 gcacctttgc cacactgagt gagctgcact gtgacaagct gcacgtggat cctgagaact
361 tcaggctcct gggcaacgtg ctggtctgtg tgctggccca tcactttggc aaagaattca
421 ccccaccagt gcaggctgcc tatcagaaag tggtggctgg tgtggctaat gccctggccc
481 acaagtatca ctaagctcgc tttcttgctg tccaatttct attaaaggtt cctttgttcc
541 ctaagtccaa ctactaaact gggggatatt atgaagggcc ttgagcatct ggattctgcc
601 taataaaaaa catttatttt cattgc
The leading UTR (black) is 50 bp long. The coding sequence starts
at bp 51 (atg—in green). The 64th codon (bp 240-242: red) codes for
a histidine (cat). A cytosine to thymine (c to t) change in the first
base of the codon alters the code to a tyrosine (tat) seen in
hemoglobin M-Saskatoon. The coding sequence of 441 bp (51 to
495—in orange) is for a 147 amino acid protein. The stop codon
(taa—in green) starts at bp 492. The trailing UTR (black) starts at
bp 495. The poly A signal sequence (red) begins at bp 602.
Beta Hemoglobin Protein Structure
Substitution seen in Hemoglobin M-Saskatoon: a tyrosine
for histidine at position 63
Conservation and Evolution A. Boxshade Diagram of the beta hemoglobin across 13 mammalian species
(gi_4504349_ref_ is the human beta globin sequence). This is a highly conserved protein in mammals (see organism
key). B. Drawtree - unrooted diagram showing the phylogeny relationships of beta hemoglobin protein. C.
Drawgram- rooted phylogenic tree of beta hemoglobin protein. Both phylogenetic maps show that human Beta
Hemoglobin is most like that of the Chimpanzee and the Bonobo our closest relatives. *Note: hemoglobin homologs
are even found in bacteria!
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Summary
•Hemoglobin M-Saskatoon is a defect in the human beta hemoglobin
protein that causes cyanosis.
•The point mutation leading to this defect is a c to t base change at
base 240 in the mRNA. This change leads to a codon change from ca-t coding a histidine to t-a-t coding for tyrosine at amino acid 63.
Histidine
Tyrosine
•The histidine is a conserved amino acid that interacts with the
heme molecule and stabilizes the ferrous ion in reduced state.
•The change of histidine to tyrosine results in oxidization of the
ferrous ion and results in an inability to bind oxygen (MetHB form)..
View of the heme pocket formed by two
helices. The iron atom (orange) has an O2
molecule (red) bound to it. The two
histidine residues are shown stabilizing the
iron atom in the center of the heme ring.
The 63rd histidine is on the right.
•This mutation is only seen in heterozygotes as the homozygote is
most likely lethal. A heterozygote individual has up to 30 to 50% of
beta hemoglobin in the MetHb form.
Wildtype human beta hemoglobin
with histidine 63
Beta hemoglobin with tyrosine
mutation at position 63
The 63rd histidine stabilizes the ferrous (Fe+2) atom in the heme ring and protects it from oxidation. During oxygen binding, the
two helices of the heme pocket expands, permitting the entrance of an O2 molecule. Upon release of the O2 molecule, the heme
pocket closes and the 63rd histidine moves closer to the iron atom and forms a bonding coordinate with it, protecting the
reduced iron from oxidation.
Hb M-Saskatoon has a weak Fe-peptide interaction due to a tyrosine for histidine substitution at position 63 of the beta chain,
which leads to persistent oxidation of the iron molecule. The oxidized iron atom remains oxidized which renders the beta chain
incapable of carrying oxygen. The image of beta hemoglobin with the tyrosine substitution at amino acid 63 shows negative
interactions (in pink). Both tyrosine and histidine are polar amino acids. Histidine is positively charged while tyrosine is a larger
neutral amino acid.
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Bibliography
1. Hematology: Clinical Principles and Applications, 3rd ed., Rodak,
Fritsma and Doig, Saunders, 2007.
2. Clinical Hematology: Principles, Procedures and Correlations, 2nd
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3. Vella F, Kamuzora H, Lehmann H, Duncan B, Harold W, A second
family with hemoglobin M Saskatoon in Saskatchewan, Clinical
Biochemistry. 1974 Jun;7(2): pp186-91.
4. Kedar PS, Nadkarni AH, Phanasgoankar S, Madkaikar M, Ghosh K,
Gorakshakar AC, Colah RB, Mohanty D., Congenital
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5 .Da-Silva SS, Sajan IS, Underwood JP 3rd., Congenital
methemoglobinemia: a rare cause of cyanosis in the newborn--a
case report. Pediatrics., 2003: 112(2): pp158-61.
•Patients with this condition have slightly higher hematocrits
(measure of percent of RBCs in the blood) than normal suggesting
that the decreased oxygen carrying capacity is compensated for by
increasing the numbers of RBCs in the blood.
6 .Stamatoyannopoulos G, Nute PE, De novo mutations producing
unstable Hbs or Hbs M. II. Direct estimates of minimum nucleotide
mutation rates in man. Human Genetics, 1982; 60(2): pp181-8.
•MetHb absorbs light at a different wave length than oxyhemoglobin. This is normally evident in the color difference between
arterial blood and venous blood. Arterial blood containing almost
100% oxy-hemoglobin is bright red. Venous blood, containing up to
75% deoxy-hemoglobin is blue. The permanent presence of high
levels of MetHb in the capillary blood (close to the skin surface) in
patients with Hb M-Saskatoon imparts a blue (cyanosis) color to the
skin and is the most obvious phenoytpe of this rare mutation in the
hemoglobin beta gene.
8. Multiple tools from National Center for Biotechnology Information
(NCBI )U.S. National Library of Medicine 8600 Rockville Pike,
Bethesda, MD 20894 http://www.ncbi.nlm.nih.gov/
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paramagnetic resonance spectra. Hemoglobin. 1980; 4(3-4): pp573-4.
9. Multiple tools from San Diego Supercomputer Center (SDSC) Biology
WorkBench. University of California, San Diego.
http://workbench.sdsc.edu/
10. Guex, N. and Peitsch, M.C. (1997) SWISS-MODEL and the SwissPdbViewer: An environment for comparative protein modeling.
Electrophoresis 18, 2714-2723. http://spdbv.vital-it.ch/