Isolation, cloning and functional characterization of porcine mannose-binding lectin.
Binding of mannose-binding lectin (MBL), a C-type lectin, and its associated serine proteases, MASP-1 and MASP-2, to cell surface carbohydrates activates the lectin complement pathway. As MBL plays an important role in innate immunity, it has been cloned and characterized in several species. While the pig may be used as a source of organs/tissues for xenotransplantation, little is known about its MBL, thus, we report the isolation of three monomeric forms of MBL from porcine serum. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie staining of reduced porcine MBL revealed the presence of three monomeric forms with approximate molecular masses of 30 000, 32 000 and 34 000. Protein sequencing identified these monomeric forms as one single protein, suggesting post-translational modification. Western blot analysis demonstrated the cross-reactivity of anti-human MBL polyclonal antibody with porcine MBL. A full-length porcine liver MBL cDNA was isolated and the predicted amino acid sequence exhibited 64.9% identity with human MBL and 50.2% and 56.7% identity with rat A and C MBL, respectively. Furthermore, Northern blot analysis demonstrated the presence of a single ( approximately 1.4-1.6 kilobase pair) transcript in porcine liver. Addition of purified porcine MBL to MBL-deficient human sera augmented N-acetylglucosamine inhibitable C3 deposition to mannan-coated plates in a dose-dependent manner. Taken together, these data demonstrate that porcine and human MBL are highly conserved, sharing structural and functional characteristics.
Agah
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Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham & Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Montalto
M C
MC
Young
K
K
Stahl
G L
GL
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D037222:Collectins; D003176:Complement C3 / Q000378:metabolism; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008351:Mannans / Q000378:metabolism; D008969:Molecular Sequence Data; D013045:Species Specificity; D013552:Swine / Q000378:metabolism*
D002352:Carrier Proteins; D037222:Collectins; D003176:Complement C3; D018076:DNA, Complementary; D037102:Lectins; D008351:Mannans