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 d omain, a short linking domain ("neck" region), and a COOH-terminal carbohyd rate recognition domain (CRD). Previous studies demonstrated that SP-D is c hemotactic 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 w as expressed in Escherichia coli and purified by affinity chromatography on maltosyl-agarose. A 17-kDa recombinant SP-D CRD was identified by two anti bodies (antisynthetic SP-D COOH-terminal and neck region peptides) but not by synthetic SP-D NH2-terminal peptide antibody. The recombinant SP-D CRD w as confirmed by amino acid sequencing. Gel-filtration analysis found that 8 4% of CRD was trimeric and the rest was monomeric. Analysis of the chemotac tic properties of the trimeric CRD demonstrated that the CRD was chemotacti c 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 s uppress the chemotactic response to fMLP. The peak chemotactic activity of the CRD is comparable to the activity of native SP-D, although a higher con centration is required for peak activity (10(-10) vs. 10(-11) M). The chemo tactic response to CRD was largely prevented by preincubation of polymorpho nuclear leukocytes with SP-D, and the response to SP-D was prevented by pre incubation with CRD. These preincubations did not affect chemotaxis to fMLP . These results suggest that trimeric CRD accounts for the chemotactic acti vity of SP-D.