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Blood, 15 May 2001, Vol. 97, No. 10, pp. 3306-3307
BRIEF REPORT
Human polymorphism of P-selectin glycoprotein ligand 1 attributable to variable numbers of tandem decameric repeats in the
mucinlike region
Vahid Afshar-Kharghan,
Reyhan Diz-Küçükkaya,
Erwin H. Ludwig,
Ali J. Marian, and
José A. López
From the Departments of Medicine and Molecular and
Human Genetics, Baylor College of Medicine, Houston, TX, and the
Gladstone Institute of Cardiovascular Disease, San Francisco,
CA.
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Abstract |
Platelet GP Ib and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1) are membrane mucins with a number of structural and functional
similarities. It was investigated whether, like GP Ib, PSGL-1 is
affected by a variable number of tandem repeat polymorphism in its
mucin-like region. By polymerase chain reaction amplification of the
genomic region encoding the PSGL-1 repeats, 3 allelic variants were
identified in the human population. The 3 alleles A, B, and Cfrom
largest to smallest, contained 16, 15, and 14 decameric repeats,
respectively, with the B variant lacking repeat 2 and the C variant
retaining repeat 2 but lacking repeats 9 and 10. Allele frequencies
were highest for the A variant and lowest for the C variant in the 2 populations studied (frequencies of 0.81, 0.17, and 0.02 in white
persons and 0.65, 0.35, and 0.00 in Japanese). Thus, PSGL-1 is highly
polymorphic and contains a structural polymorphism that potentially
indicates functional variation in the human population.
(Blood. 2001;97:3306-3307)
© 2001 by The American Society of Hematology.
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Introduction |
Platelet glycoprotein (GP) Ib and leukocyte
P-selectin glycoprotein ligand 1 (PSGL-1) have a number of interesting
similarities. Both mediate the attachment of blood cells from the bulk
flow onto the blood vessel wallGP Ib attaching platelets to the
subendothelium through binding von Willebrand factor
(vWF)1 and PSGL-1 tethering leukocytes to P-selectin on
the surface of activated endothelium.2 Both also mediate
rolling interactions over surfaces coated with their respective
ligands.3,4 To tether cells to these surfaces, each
receptor has independently evolved a mucin-like stalk that allows the
ligand-binding region to be extended above the forest of other proteins
on the plasma membrane. Both stalks have arisen by similar mechanisms:
tandem repetition of short amino acid stretches containing threonine,
serine, and proline within heavily glycosylated, mucin-like sequences.
The basic repeated unit in GP Ib comprises 13 amino acids, and in
PSGL-1 it contains 10 amino acids. In human populations, GP Ib is
polymorphic based on variable numbers of these tandem repeats
(VNTRs),5 a polymorphism that has been shown to be a
marker for increased susceptibility to cardiovascular disease.6 We hypothesized that PSGL-1 could similarly be
affected by a VNTR polymorphism.
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Study design |
Study population
All subjects signed informed consent documents, and the
institutional review board of the Baylor College of Medicine approved the protocols. Two independent populations were studied, the first comprising 141 unrelated persons in the CEPH (Centre d'Etude du Polymorphisme Humain) population and the second comprising 68 apparently healthy Japanese blood donors.
Genotyping
A set of oligonucleotide primers was designed to encompass the
decameric consensus repeat sequences in exon 2 of the PSGL-1 gene. The sequences of the forward and reverse primers were
5'-CCTGTCCACGGATTCAGC-3' and 5'-GGGAATGCCCTTGTGAGTAA-3' (corresponding
to nucleotide sequences 576-593 and 1134-1115, respectively).7 DNA was amplified by polymerase chain
reaction (PCR) using 100 ng DNA, 200 nM each primer, 200 µM each
dNTP, 1.0 µM MgCl2, and 1.5 U Taq DNA
polymerase for 35 cycles. The melting temperature was 94° C for 30 seconds, annealing temperature was 62°C for 45 seconds, and the
extension temperature was 72°C for 90 seconds. PCR products
were separated on 2% agarose gels and were visualized by ethidium
bromide staining. PCR products from homozygous persons were sequenced.
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Results and discussion |
Identification, frequency, and molecular basis of a PSGL-1 VNTR
polymorphism
Genotyping was successfully completed in all 141 subjects in the
CEPH population and 68 Japanese blood donors. Gel electrophoresis of
the PCR products identified 3 bands558 bp, 528 bp, and 498 bp (not
shown)which were labeled as alleles A, B, and C, respectively. Sequence analysis showed that the A allele was identical to the form
stored in the GenBank database (accession number U25956), the B allele
lacked the second tandem repeat (ATEAQTTQPV), and allele C retained the
second tandem repeat but lacked repeats 9 and 10 (AMEAQTTAPA and
AMEAQTTPPA, respectively) (Figure 1). Allele and genotype frequencies are shown in Table
1. For these studies, the
PSGL-1 genes were sequenced from 2 AA homozygotes, 1 BB
homozygote, 1 CC homozygote, and 1 BC heterozygote. Thus, 4 A alleles
and 3 each of the B and C alleles were sequenced. Although only 3 PCR
products were detected in 209 subjects, it remains possible that other
alleles exist of the same length but lack other repeats or that the
polypeptide is also polymorphic at the level of single amino
acid changes.
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| Figure 1.
Schematic depiction of the 3 PSGL-1 polymorphic
variants.
Each decameric repeat is depicted as an oval. The largest variant (A)
has 16 repeats, the B variant has 15 repeats (repeat 2 of the A form is
missing), and the C variant has 14 repeats (repeats 9 and 10 of the A
form are missing). Repeats of the A variant missing in the smaller
variants are shaded gray; their sequences are given below the
A variant.
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This polymorphism thus bears a striking analogy to the VNTR
polymorphism of the GP Ib gene.5,8 Based on
analogy to mucins for which physicochemical studies have been carried
out, it has been predicted that each added amino acid in the mucin-like
region of GP Ib adds 2.5 Å to the length of the macroglycopeptide
stalk, with the addition of three 13-amino acid repeats adding
approximately 100 Å to the length of the region.5 With
PSGL-1, the difference between the shortest and longest form would not
be expected to be as great (approximately 50 Å). Nevertheless, this
difference may have an even greater effect on the actual receptor
because PSGL-1 forms disulfide-linked dimers and heterozygosity could lead to a disparity in the spatial relationship of the ligand-binding sites on the individual polypeptides of the dimer. One study has suggested that PSGL-1 dimerization is required for the interaction of
PSGL-1 with P-selectin,9 but this requirement was not
supported by another study.4 Thus, either by changing the
distance of important ligand-binding regions from the leukocyte plasma
membrane or by affecting the relationship between ligand-binding
regions on adjacent polypeptide chains (in a heterozygous person), the PSGL-1 polymorphism is likely to subtly affect binding to P-selectin and, therefore, leukocyte function.
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Footnotes |
Submitted November 29, 2000; accepted January 19, 2001.
Supported by National Institutes of Health grants HL54218, 9-640-133N,
and 96-012-670. J.A.L. and A.J.M. are Established Investigators of the
American Heart Association.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: José A. López, Department of Medicine,
Baylor College of Medicine, BCM286, N1319, One Baylor Plaza, Houston,
TX 77030; e-mail: josel{at}bcm.tmc.edu.
| |
References |
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Fredrickson BJ, Dong JF, McIntire LV, López JA.
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J Biol Chem.
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Ishida F, Furihata K, Ishida K, et al.
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Abstract
Introduction