Detection of Human PSGL‑1/CD162 by Western Blot. Western blot shows lysates of human peripheral blood lymphocytes (PBL). PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human|
PSGL‑1/CD162 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3345) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for PSGL‑1/CD162 at approximately 115‑120 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8.
Human PSGL-1 (P-Selectin Glycoprotein Ligand-1; also CD162), is a 120 kDa mucin-type glycoprotein that plays a key role in leukocyte adhesion (1-3). It is synthesized as a 412 amino acid (aa) preproprecursor that contains a 17 aa signal sequence, a 24 aa propeptide, a 279 aa extracellular domain (ECD), a 21 aa transmembrane segment and a 71 aa cytoplasmic region (4, 5). Following cleavage of the pre- and prosegments, it is expressed as a 240 kDa disulfide-linked homodimer. The extreme N-terminus (aa 1-16 of the mature molecule) contains one threonine (#16) and three tyrosines (#5, 7, and 10) that are involved in ligand binding. The Thr residue allows for O-linked glycosylation in the form of a core-2 structure (GalNAc-Gal) linked in a beta 1,6 bond to a sialylated Lewis X motif (GlcNAc linked to both Fuc and Gal with a terminal sialic acid residue) (1, 2, 5, 6, 7). The three tyrosine residues allow for sulfation (8, 9). When binding to P-selectin, Tyr sulfation and glycosylation are essential. Tyr7 provides the most efficient sulfate moiety, while Fuc and sialic acid are essentially mandatory (7). When binding to
E‑Selectin, only carbohydrate is needed, while both carbohydrate and Tyr10 are used for L-selectin binding (6, 8). There are 16 decameric aa repeats in the ECD of the longform of PSGL-1. This form is referred to as the A allele, and represents 65-80% of the population. Alleles B and C show deletions of decameric repeats #2 (aa 132‑141) plus #9 and 10 (aa 222-241), respectively. Shorter forms may show weaker binding to P-selectin (9, 10). Soluble forms of PSGL-1 are also known. Neutrophil elastase will cleave somewhere within repeats #5-9, while cathepsin G cleaves after Tyr7 (11). The loss of Tyr5 and 7 should impact binding affinity.
PSGL‑1 is found on virtually all leukocytes and macrophages/DC’s (1). Although there is similarity in the organization of the ECD between species, there is little aa identity. Human PSGL-1 ECD is 51%, 52% and 43% aa identical to equine, canine and mouse ECD, respectively.
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