Asok Chaudhuri

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression.

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.

A flexible array format for large‐scale, rapid blood group DNA typing

BACKGROUND: Typing for blood group antigens is currently performed by hemagglutination. The necessary reagents are becoming costly and limited in availability, and the methods are labor‐intensive. The purpose of this study was to determine the feasibility of the use of large‐scale DNA analysis in a microarray as a substitute for blood group typing.

Deletion of the Murine Duffy Gene (Dfy) Reveals that the Duffy Receptor Is Functionally Redundant

ABSTRACT All of the antigenic determinants of the Duffy blood group system are in a glycoprotein (gp-Fy), which is encoded by a single-copy gene (FY) located on chromosome 1. gp-Fy is also produced in several cell types, including endothelial cells of capillary and postcapillary venules, the epithelial cell of kidney collecting ducts, lung alveoli, and the Purkinje cells of the cerebellum. This protein, which spans the cell membrane seven times, is a member of the superfamily of chemokine receptors and a malarial parasite receptor. The mouse Duffy gene (Dfy) homolog of human FYis also a single-copy gene, which maps in a region of conserved synteny with FY and produces a glycoprotein with 60% homology to the human protein. The mouse Duffy-like protein also binds chemokines. To study the biological role of gp-Fy, we generated a mouse strain in which Dfy was deleted. These homozygousDfy −/− mice were indistinguishable in size, development, and health from wild-type and heterozygous littermates. We also examined components of the immune system and found no differences in lymph nodes or peripheral blood leukocyte levels between knockout and wild-type mice. The gross and histological anatomy of the thymus, spleen, lung, and brain showed no significant differences between mutants and wild-type mice. There was no indication of an overall difference between the knockout and wild-type mice in systematic neurological examinations. The only significant difference betweenDfy −/− and Dfy +/+mice that we found was in neutrophil migration in peritoneal inflammations induced by lipopolysaccharide and thioglycolate. In mice homozygous for the deletion, there was less neutrophil recruitment into the peritoneal cavity and neutrophil influx in the intestines and lungs than in wild-type mice. Despite this, the susceptibility toStaphylococcus aureus infection was the same in the absence and in the presence of gp-Fy. Our results indicate that gp-Fy is functionally a redundant protein that may participate in the neutrophil migratory process.

The Duffy protein: a malarial and chemokine receptor.

A major advance towards understanding the Duffy blood group system has been achieved with the cloning of FY, a single-copy gene located in the 1q22->q23 region of chromosome 1. The product of FY Is an acidic glycoprotein (gp-Fy), which spans the plasma membrane seven times and has an exocellular N-terminal domain and an endocellular C-terminal domain. The system consists of four alleles, five phenotypes, and five antigens. FYA, FYB, FYB(ES), and FYB(WK) are the alleles; Fy(a+b-), Fy(a-b+), Fy(a+b+), Fy(a-b+(wK)), and Fy(a-b-), are the phenotypes, and Fy(a), Fy(b), Fy3, Fy5, and Fy6 are the antigens. Fy(a-b-), or Duffy-negative individuals, lack the Duffy protein on erythrocytes and are predominantly African and American blacks. They have the FYB(Es) allele with a mutation in the promoter region, which abolishes the expression of the protein in erythrocytes only. In the few cases of non-black Fy(a-b-) individuals, a nonsense mutation prevents the synthesis of gp-Fy. In Fy(a-b+(wk)) erythrocytes, the Fy(b) antigen is weakly expressed due to a reduced amount of the protein. The Fy5 antigen includes the Rh protein, and the Fy6 antigen is defined by a murine monoclonal antibody. Gp-Fy is produced in several cell types, including endothellal cells of capillary and postcapillary venules, epithelial cells of kidney collecting ducts, and lung alveoli, as well as PurkinJe cells of the cerebellum. The Duffy protein plays a role in inflammation and in malaria Infection. The protein is a member of the superfamily of chemokine receptors and is the receptor to which certain malarial parasites bind to invade red blood cells. The parasite-specific binding site, the binding site of chemokines, and the major antigenic domains are located in overlapping regions at the exocellular N terminus of the Duffy protein.

Molecular mechanisms that lead to reduced expression of Duffy antigens.

BACKGROUND: In the Duffy blood group system, the null phenotype Fy(a–b–) has been classically associated with a mutated GATA box, while the Fyx phenotype weak Fyb is associated with Arg89Cys and Ala100Thr mutations. This report assesses the prevalence of the Duffy GATA box and the Fyx‐associated mutations in white and African American (black) donors and investigates the molecular mechanism underlying the Fyx phenotype.

Detection of blood group genes using multiplex SNaPshot method

BACKGROUND: The determination of blood group antigens in patients and donors is of primary importance in transfusion medicine. Blood group antigens are inherited and are polymorphic in nature. The majority of polymorphic blood group antigens arise from single‐nucleotide polymorphisms (SNPs) in the blood group genes. Many DNA‐based assays, such as species‐specific polymerase chain reaction (PCR), PCR–restriction fragment length polymorphism, and microchips, have been described to study variant blood group genes. In this study, the SNaPshot (Applied Biosystems) method was adapted to detect SNPs in 10 common blood group systems.

The production of red blood cell alloantibodies in mice transfused with blood from transgenic Fyb‐expressing mice

BACKGROUND: A murine model would be useful to identify which immune mechanisms could be manipulated to treat or prevent red blood cell (RBC) alloimmunization in patients who become sensitized to multiple or widely expressed antigens.

The Fya, Fy6 and Fy3 epitopes of the Duffy blood group system recognized by new monoclonal antibodies: identification of a linear Fy3 epitope.

Four new anti‐Duffy murine monoclonal antibodies (MAbs): two anti‐Fy6 (MIMA‐107 and MIMA‐108), one anti‐Fya (MIMA‐19) and one anti‐Fy3 (MIMA‐29) were characterized. Identification of epitopes by means of synthetic peptides (Pepscan) showed that the anti‐Fy6 reacted most strongly with peptides containing the sequence 19QLDFEDV25 of the Duffy glycoprotein, and less strongly with peptides containing LDFEDV (MIMA‐107) or LDF only (MIMA‐108). The anti‐Fya recognized epitope 38DGDYGA43 containing the Gly42 residue, which defines the Fya blood group antigen. MIMA‐29 is the first anti‐Fy3 reactive with a linear epitope 281ALDLL285 located in the fourth extracellular domain (ECD4, loop 3) of the Duffy glycoprotein. The four new antibodies extend the list of six anti‐Fy MAbs formerly characterized by Pepscan analysis that allow some general conclusions. Fine specificities of various anti‐Fya, or anti‐Fy6 are not identical, but all of them recognize linear epitopes located around, respectively, Gly42 or between two potential N‐glycosylation sites at Asn16 and Asn27. Anti‐Fy3 recognize either a linear epitope located in ECD4, or a conformational epitope that includes amino acid residues of ECD4 and of other ECDs.

Use of RhD fusion protein expressed on K562 cell surface in the study of molecular basis for D antigenic epitopes

The human D antigens, one of the most clinically important blood groups, are presented by RhD protein with a putative 12 transmembrane topology. To understand the molecular basis for the complex antigenic profile of RhD protein, we expressed a series of RhD fusion proteins using different portions of Duffy protein as a tag in erythroleukemic K562 cells. Because the reactivity of monoclonal anti-RhD antibody, LOR15C9, depends mainly on the sequence coded by exon 7 of RhD, we altered DNA sequence corresponding to the amino acid residues 323–331(A) and 350–354(B) in the exon 7. The mutation in region B resulted in a severe reduction in LOR15C9 binding by flow cytometry analysis, suggesting that region B may play an important role in constituting antigen epitopes recognized by LOR15C9. On the other hand, a slight decrease in the antibody binding was observed for the region A mutant, suggesting that the intracellularly located region A may elicit a long distance effect on the formation of exofacial antigen epitopes. In addition, using various monoclonal antibodies against RhD, we compared the antigenic profile of expressed RhD fusion protein with that of endogenous RhD in K562 cells as well as in erythrocytes.

Immunization of transgenic mice for production of MoAbs directed at polymorphic blood group antigens

BACKGROUND: Antibodies of human origin for blood typing are increasingly difficult to obtain, and, despite aggressive efforts, MoAbs with specificities to several blood group polymorphisms have eluded production. As an approach for the generation of MoAbs with defined specificities, the feasibility of immunizing mice that are transgenic for the target polymorphism, Fya/Fyb of the Duffy blood group system, was tested with a source of the antithetical antigen.