Binding of human milk to pathogen receptor DC-SIGN varies with bile salt-stimulated lipase (BSSL) gene polymorphism

Abstract

Objective: Dendritic cells bind an array of antigens and DC-SIGN has been postulated to act as a receptor for mucosal pathogen transmission. Bile salt-stimulated lipase (BSSL) from human milk potently binds DC-SIGN and blocks DC-SIGN mediated trans-infection of CD4(+) T-lymphocytes with HIV-1. Objective was to study variation in DC-SIGN binding properties and the relation between DC-SIGN binding capacity of milk and BSSL gene polymorphisms.

Study design: ELISA and PCR were used to study DC-SIGN binding properties and BSSL exon 11 size variation for human milk derived from 269 different mothers distributed over 4 geographical regions.

Results: DC-SIGN binding properties were highly variable for milks derived from different mothers and between samplings from different geographical regions. Differences in DC-SIGN binding were correlated with a genetic polymorphism in BSSL which is related to the number of 11 amino acid repeats at the C-terminus of the protein.

Conclusion: The observed variation in DC-SIGN binding properties among milk samples may have implications for the risk of mucosal transmission of pathogens during breastfeeding.

Figure 1. DC-SIGN binding is highly variable and correlates with BSSL protein size.

(a) Example of a coomassie stained SDS-PAGE separation of breast milk from 7 mothers showing the bile salt-stimulated lipase (BSSL) of variable sizes. (b) Western blot stained with DC-SIGN-Fc of the same 7 milks as depicted in figure a. (c) Breast milks with smaller BSSL protein have stronger DC-SIGN binding capacity than breast milks with larger BSSL protein. Molecular weights (MW) of BSSL protein was compared in milks with strong DC-SIGN binding capacity versus milks with weak DC-SIGN binding capacity. Median protein sizes in the weak and in the strong DC-SIGN binding groups are indicated by a horizontal line.

Figure 2. DC-SIGN affinity of breast milk is highly variable between mothers within cohorts and between different cohorts.

The DC-SIGN binding of milks from the Netherlands (NL), Sweden (SW), Norway (NO) and Egypt (EG) is depicted as a percentage of the highest overall binding sample (100%). Median DC-SIGN affinity is indicated by a horizontal line for each cohort.

Figure 3. BSSL exon 11 is highly polymorphic in number of repeats.

(a) Typical agarose gel analysis of BSSL exon 11 PCR genotyping including 3 marker lanes and 7 genotyped DNA samples. (b) Allelic repeat number variation as determined by PCR analysis within each cohort. Repeat number frequency is indicated as percentage of the total number of alleles within a cohort. (c) BSSL genotype distribution is highly variable in all cohorts with most mothers having at least 1 allele with 16 repeats. Overview is plotted of the BSSL repeat number genotype combinations per milk sample within each cohort.

Figure 4. DC-SIGN binding capacity correlates to BSSL genotype.

Alleles are defined as L (9 to 15 repeats) or H (16–19 repeats). The BSSL genotype is plotted against DC-SIGN affinity for mothers from the Netherlands, Sweden, Norway and Egypt. LL, LH and HH genotypes are plotted against DC-SIGN binding. Median DC-SIGN binding within cohorts is indicated by a horizontal line.