Recombinant SP-D carbohydrate recognition domain is a chemoattractant for human neutrophils.
Human pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin with high binding specificity to alpha-D-glucosyl residues. It is composed of four regions: a short NH2-terminal noncollagen sequence, a collagenous domain, a short linking domain ("neck" region), and a COOH-terminal carbohydrate recognition domain (CRD). Previous studies demonstrated that SP-D is chemotactic for inflammatory cells. To test which domain of SP-D might play a role in this function, a mutant that contains only neck and CRD regions was expressed in Escherichia coli and purified by affinity chromatography on maltosyl-agarose. A 17-kDa recombinant SP-D CRD was identified by two antibodies (antisynthetic SP-D COOH-terminal and neck region peptides) but not by synthetic SP-D NH2-terminal peptide antibody. The recombinant SP-D CRD was confirmed by amino acid sequencing. Gel-filtration analysis found that 84% of CRD was trimeric and the rest was monomeric. Analysis of the chemotactic properties of the trimeric CRD demonstrated that the CRD was chemotactic for neutrophils (polymorphonuclear leukocytes), with peak activity at 10(-10) M equal to the positive control [formyl-Met-Leu-Phe (fMLP) at 10(-8) M]. The chemotactic activity was abolished by 20 mM maltose, which did not suppress the chemotactic response to fMLP. The peak chemotactic activity of the CRD is comparable to the activity of native SP-D, although a higher concentration is required for peak activity (10(-10) vs. 10(-11) M). The chemotactic response to CRD was largely prevented by preincubation of polymorphonuclear leukocytes with SP-D, and the response to SP-D was prevented by preincubation with CRD. These preincubations did not affect chemotaxis to fMLP. These results suggest that trimeric CRD accounts for the chemotactic activity of SP-D.
Cai
G Z
GZ
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Griffin
G L
GL
Senior
R M
RM
Longmore
W J
WJ
Moxley
M A
MA
D002630:Chemotactic Factors / Q000235:genetics* / Q000494:pharmacology*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D006801:Humans; D009154:Mutation / Q000235:genetics; D009504:Neutrophils / Q000187:drug effects*; D011489:Protein Denaturation; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D011994:Recombinant Proteins
D002630:Chemotactic Factors; D006023:Glycoproteins; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants; D011994:Recombinant Proteins