Bile salt-stimulated lipase from human milk binds DC-SIGN and inhibits human immunodeficiency virus type 1 transfer to CD4+ T cells

Abstract

A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium, Leishmania, and Helicobacter pylori, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4+ T lymphocytes in trans and enhance infection. We have previously demonstrated that a multimeric LeX component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (LeX)-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4+ T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in LeX expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents.

FIG. 1.

Biochemical analysis of DC-SIGN binding component in human milk. (A) The DC-SIGN-Fc binding capacity of trypsin-treated human milk (HM) (end concentration, 1/40) was measured by DC-SIGN-Fc binding ELISA. The DC-SIGN-Fc binding background level was obtained by preincubation with AZN-D1 (DC-SIGN-specific blocking antibody) and EGTA. * represents a P value of <0.01 compared to the binding of the human milk incubated with RPMI. (B) Raji-DC-SIGN cells were incubated with the trypsin-treated human milk or controls in the presence of HIV-1 before being washed and before CD4⁺ T lymphocytes were added. As a control, Raji or Raji-DC-SIGN cells were incubated with PBS and virus before the addition of CD4⁺ T lymphocytes. CA-p24 production was measured at day 7 by standard ELISA. * represents a P value of <0.01 for inhibition compared to that of Raji-DC-SIGN. (C) Human milk (dilution, 1:2) was heated at 99°C for 10 min before the determination of the DC-SIGN-Fc binding capacity. AZN-D1 and EGTA were used as controls to show DC-SIGN-Fc-specific binding. * represents a P value of <0.001 in comparing heat-treated milk to a nontreated sample. Standard deviations are depicted in all graphs.

FIG. 2.

The inhibitory component of human milk is present in the >100-kDa fraction of human milk. (A) Levels of DC-SIGN-Fc binding of the differently sized fractionations were determined for the human milk fractions (1:100) in the DC-SIGN-Fc binding ELISA, with AZN-D1 and EGTA controlling for DC-SIGN binding specificity. * represents a P value of <0.01 compared to the relevant control. Standard deviations are depicted. (B) Raji-DC-SIGN cells were incubated with the differently sized fractions before being washed and before the addition of fluorescent gp120 beads. To control for DC-SIGN-specific binding, the cells were also incubated with EGTA, mannan, and AZN-D1. HM, human milk. (C) The Raji-DC-SIGN cells were incubated with the different human milk size fractions (1/4) and virus before the addition of CD4⁺ T lymphocytes. As a control, Raji or Raji-DC-SIGN cells were incubated with PBS and virus before the addition of CD4⁺ T lymphocytes. CA-p24 production was measured at day 7 by standard ELISA. * represents a P value of <0.01 compared to the PBS control. Standard deviations are depicted.

FIG. 3.

Differences in the DC-SIGN binding capacities of human milk samples from three mothers (S1 to S3). (A) The DC-SIGN-Fc binding capacity was measured for three different human milk samples (1:200). Preincubation of DC-SIGN-Fc with AZN-D1 and EGTA controlled for DC-SIGN-specific binding. * represents a P value of <0.01 compared to normal DC-SIGN-Fc binding. (B) Different dilutions of the milk samples (S1 to S3) were tested in the Raji-DC-SIGN transfer culture assay. To control for infection, Raji or Raji-DC-SIGN cells were incubated with PBS and virus before the addition of CD4⁺ T lymphocytes. CA-p24 production was determined on day 7 by standard ELISA. * represents a P value of <0.02 compared to the PBS control. Standard deviations are depicted in both graphs.

FIG. 4.

Western blotting and Coomassie staining of three human milk samples with different DC-SIGN binding capacities. (A) Western blot of human milk samples S1, S2, and S3 stained with Le^X-specific antibody. (B) Western blot stained with DC-SIGN-Fc. (C) Coomassie-stained SDS-PAGE gel. Molecular weights are indicated to the left.

FIG. 5.

BSSL binds DC-SIGN and prevents the transfer of HIV-1 to CD4⁺ T lymphocytes. (A) Raji-DC-SIGN cells were incubated with different dilutions of native BSSL (nBSSL) isolated from human milk (S4) and virus before the addition of CD4⁺ T lymphocytes. As a control, Raji and Raji-DC-SIGN cells were incubated with PBS instead of BSSL. CA-p24 production was determined on day 7 by standard ELISA. * represents a P value of <0.05 compared to the PBS control. (B) DC-SIGN-Fc binding capacity was determined by ELISA for different dilutions of BSSL isolated from human milk (S4). To control for DC-SIGN specificity, DC-SIGN-Fc was preincubated with AZN-D1 and EGTA to allow comparison to the relevant binding without inhibitor. * represents a P value of <0.01. Standard deviations are depicted in both graphs.

FIG. 6.

DC-SIGN-Fc binding can be blocked with a Le^X-specific antibody. BSSL was plated at a concentration of 0.3 μg/ml, and before the addition of DC-SIGN-Fc, the coated BSSL was preincubated with the Le^X-specific antibody. DC-SIGN-Fc was also preincubated with AZN-D1 and EGTA to determine the specificity of binding.

FIG. 7.

Variant BSSL isoforms of different sizes show differential binding to DC-SIGN. Two BSSL isoforms isolated from the same human milk sample (S5) demonstrate a difference in DC-SIGN-Fc binding capacity. Different dilutions of the BSSL were plated, and the DC-SIGN-Fc binding was determined by ELISA. To control for the specificity of the binding, DC-SIGN-Fc was preincubated with AZN-D1 or EGTA. The highest binding value observed for these controls was subtracted from the observed value for binding without an inhibitor.