Molecular identification of PAL-E, a widely used endothelial-cell marker

Abstract

The pathologische anatomie Leidenendothelium (PAL-E) antibody has been used for almost 20 years as a specific marker for vascular endothelial cells. Due to the fact that this antibody works only in very limited applications, the molecular identity of PAL-E has remained unknown. IntroductionSeveral antigens including factor VIII, CD31, CD36, ulex europaeus lectin, and pathologische anatomie Leiden-endothelium (PAL-E) have been used as endothelial cell markers in a vast number of publications. However, none of these markers is perfect, because they either do not stain all types of endothelia or are not specific for vasculature. PAL-E has been considered a good marker because it discriminates blood vessel endothelium from lymphatic endothelium and does not stain other cell types. 1-3 Despite the relatively wide use of PAL-E as an endothelial marker, its molecular identity has remained an enigma, most likely due to the limited applicability of the antibody against the PAL-E molecule. However, a recent report suggests that the PAL-E antigen is a secreted form of vimentin. 4 The molecule defined by PAL-E is widely expressed on vasculature in different organs except in the brain. However, its expression is induced in brain tumors simultaneously with the loss of the blood-brain barrier. 5 Expression of PAL-E antigen is also increased on endocardium of rejected human cardiac allografts. 6 Plasmalemmal vesicle 1 (PV-1) has been described as a glycoprotein associated to plasmalemmal vesicles (caveolae). It is a 60-type II transmembrane protein that tends to form homodimers. Its cellular localization in the rat has been carefully analyzed both at light and electron microscopic levels. 7,8 However, the function of PV-1 has remained unknown. Expression of rat PV-1 is developmentally regulated in testis and it binds heparin. 9 The protein sequences of rat and mouse PV-1 have phosphorylation site(s) for casein kinase II, but this site is lacking in the human sequence. 10 The corresponding human sequence does not bear significant homology to any other known proteins, and, therefore, the sequence information has not helped in predicting the function of this molecule. 10 Our original aim was to identify new endothelial molecules involved in leukocyte trafficking. For this purpose, we raised monoclonal antibodies (mAbs) against small isolated vessels from the hilus of human lymph nodes. The expression pattern of one antibody showed striking specificity for endothelial cells, and, therefore, the antigen recognized by this antibody was selected for further characterization. Tryptic peptides and subsequent analyses of its cDNA showed homology of this antigen to rat PV-1 cDNA and a human cDNA designated fenestrated endothelial-linked structure protein (FELS). On the other hand, the similarities in the staining patterns between our antibody and PAL-E led to the present studies elucidating the identity of the molecule recognized by PAL-E. Materials and methods Production of the monoclonal antibodyBalb/C mice were immunized by injecting ...

PV-1 is recognized by the PAL-E antibody and forms complexes with NRP-1

“…Despite its widespread use, the identification of the PAL-E antigen has proven difficult, and there has been marked confusion regarding the identity of the molecule recognized by PAL-E. [2][3][4] We identified the molecular target of PAL-E as plasmalemma vesicleassociated protein-1 (PV-1). 3,5 Recently, this was challenged by reporting neuropilin-1 (NRP-1) as the antigen for PAL-E. 4 PV-1 is a heavily glycosylated approximately 55-to 65-kDa glycoprotein originally discovered in rat lung endothelium and subsequently found in stomatal diaphragms of endothelial caveolae and transendothelial channels.…”

“…3,5 Recently, this was challenged by reporting neuropilin-1 (NRP-1) as the antigen for PAL-E. 4 PV-1 is a heavily glycosylated approximately 55-to 65-kDa glycoprotein originally discovered in rat lung endothelium and subsequently found in stomatal diaphragms of endothelial caveolae and transendothelial channels. 6 NRP-1, on the other hand, was first found as a semaphorin receptor and neuronal cell guidance molecule 7 and later also shown to function during angiogenesis as a VEGFR-2 coreceptor binding VEGF-A 165 .…”

The Original Pathologische Anatomie Leiden-Endothelium Monoclonal Antibody Recognizes a Vascular Endothelial Growth Factor–Binding Site within Neuropilin-1

“…However, the full understanding of the interaction attributes among the receptor, corresponding known native ligands, synthetic peptides, and PAL-E will have to await elucidation of X-ray crystal structures of the protein complexes. Of course, one cannot entirely rule out the possibility that the true PAL-E antibody may recognize more than one antigen (12,13), perhaps in different settings. Indeed, cross-reaction of monoclonal antibodies with multiple antigens has long been shown (35,36).…”

“…Next, based on immunostaining and biochemical experiments, the authors have surmised that 174/2 acts as a ''PAL-E-like'' monoclonal antibody; as such, they have plausibly proposed PV-1/FELS as another PAL-E candidate antigen (13). However, neither monoclonal antibody inhibits the other in competitive staining experiments (13), bringing such data interpretation into question.…”

“…On the other hand, Niemelä et al (13) have shown that the plasmalemmal vesicle 1/fenestrated endothelial-linked structure (PV-1/FELS) protein is the antigen recognized by 174/2, a monoclonal antibody generated by immunizing BALB/c mice with vessels from human lymph nodes and fusing lymphocytes with myeloma cells. Next, based on immunostaining and biochemical experiments, the authors have surmised that 174/2 acts as a ''PAL-E-like'' monoclonal antibody; as such, they have plausibly proposed PV-1/FELS as another PAL-E candidate antigen (13).…”