Isolation, Characterization, and Antimicrobial Properties of Bovine Oligosaccharide-binding Protein

Abstract

Peptidoglycan recognition proteins (PGRPs) constitute a recently characterized family of pattern-recognition molecules that are conserved from insects to humans and are implicated in mammalian innate immunity. Here we report the isolation, characterization, cDNA cloning, and antimicrobial activities of a bovine PGRP ortholog termed bovine oligosaccharidebinding protein (bOBP). Milligram quantities of bOBP were purified from peripheral leukocytes, thus allowing for the characterization of the disulfide array and for determining the in vitro antimicrobial activities of the native protein. Of the tissues analyzed, bOBP mRNA was detected only in bone marrow where the protein is synthesized as a 190 amino acid precursor. The mature 169 amino acid protein is stored in the cytoplasmic granules of neutrophils and eosinophils but is absent from lymphocytes, monocytes, and platelets. bOBP was microbicidal for Gram-positive and Gram-negative bacteria and yeast at low micromolar concentrations. The finding that bOBP was microbicidal for organisms in which peptidoglycan is absent (Cryptococcus neoformans) or buried (Salmonella typhimurium) indicates that previous conclusions about the specificity of peptidoglycan recognition proteins must be reevaluated and suggests that other envelope components may mediate the antimicrobial action of PGRP family members.Studies over the past two decades have revealed that antimicrobial innate immunity plays a critical role in host defense mediated in part by pattern-recognition proteins and microbicidal effector molecules that contain infection prior to an adaptive immune response (1). Pattern-recognition proteins distinguish invading microbes by identifying conserved microbial structures that are not expressed by host cells (2). Microbial killing is mediated by granulocytes and macrophages that produce oxygen-and nitrogen-derived toxins (3, 4) as well as microbicidal proteins and peptides (1).Among the pattern-recognition proteins of innate immunity are lipopolysaccharide (LPS) 1 -binding protein and bactericidal/ permeability-increasing protein, molecules that bind LPS by amino-terminal structures conserved between the two proteins (5, 6). Bactericidal/permeability-increasing protein is potently bactericidal for Gram-negative bacteria (7,8) and neutralizes the inflammatory properties of LPS (9). In contrast, LPS-binding protein is not bactericidal and activates an inflammatory response in myeloid cells upon LPS binding (10, 11). Toll-like receptors (TLRs) conserved from insects to humans (12) are key elements of innate immunity (13,14 Peptidoglycan recognition proteins (PGRPs) are recently characterized components of innate immunity, which are reported to bind specifically to peptidoglycan moieties. These studies were the basis for concluding that PGRPs selectively contribute to innate immunity by detecting 25). Silkworm PGRP, the first member of the family, was implicated in the innate immune response by its ability to trigger the prophenoloxidase cascade in the presence of peptidoglyca...

Peptidoglycan Recognition Proteins Involved in 1,3-β-D-Glucan-dependent Prophenoloxidase Activation System of Insect

“…Hd-PGRP-1 and Hd-PGRP-2 were detected by immunoblotting with affinity-purified antibodies against Hd-PGRP-1 and Hd-PGRP-2. (37). The closed circles and closed reverse triangles indicate the conserved cysteine and tryptophan residues, respectively.…”

“…As shown in Fig. 3, the homology between the Hd-PGRP-1, Hd-PGRP-2, and those in the National Center for Biotechnology Information data base showed that Hd-PGRP-1 is similar to Bombyx mori PGRP (Bm-PGRP, 42.2% identity) (9), Trichoplusia ni PGRP (Tn-PGRP, 43.1%) (8), Drosophila melanogaster PGRP-SA (Dm-PGRP, 45.7%) (35), human PGRP (39.1%) (8), mouse PGRP (40.2%) (8), rat PGRP (37.0%) (36), and bovine oligosaccharide-binding protein (bovine OBP, 39.2%) (37). Four cysteine and four tryptophan residues of Hd-PGRP-1 are conserved as in all insect PGRPs as well as in human (indicated by closed circles and closed reverse triangles in Fig.…”

Gene transfer approaches in cancer immunotherapy

“…72,73 Mammalian Tag7/PGRP-S has been cloned from mouse, 69 human, 70 rat, 74 and cow. 75 There are small extracellular proteins (19)(20)(21)(22)(23)(24), which structurally share homology with T phage lyzozyme but do not possess any amidaze activity. Apart from their ability to recognize oligosaccharides and particularly peptidoglycan 70,75 cytotoxicity towards mammalian cells, 69,75,76 bactericidal and bacteriostatic activities 75 were detected.…”

“…75 There are small extracellular proteins (19)(20)(21)(22)(23)(24), which structurally share homology with T phage lyzozyme but do not possess any amidaze activity. Apart from their ability to recognize oligosaccharides and particularly peptidoglycan 70,75 cytotoxicity towards mammalian cells, 69,75,76 bactericidal and bacteriostatic activities 75 were detected. Tag7/PGRP-S is expressed in the spleen, brain, intestine, 69,74,77 bone marrow, 70 in lymphocytes 78 and neutrophils.…”

“…Tag7/PGRP-S is expressed in the spleen, brain, intestine, 69,74,77 bone marrow, 70 in lymphocytes 78 and neutrophils. 70,75 Isoforms of mammalian TagL/PGRP-L are rather large proteins, which share strong C-terminal homology with Tag7/PGRP-S but also have transmembrane domain and intracellular N terminus. 71,73 TagL/ PGRP-L was found to be both secreted and membranebound proteins recognizing a wide variety of pathogens independently of the surface peptidoglycan.…”