The scavenging capacity of DMBT1 is impaired by germline deletions

Abstract

The Scavenger Receptor Cysteine-Rich (SRCR) proteins are an archaic group of proteins characterized by the presence of multiple SRCR domains. They are membrane-bound or secreted proteins, which are generally related to host defense systems in animals. Deleted in Malignant Brain Tumors 1 (DMBT1) is a SRCR protein which is secreted in mucosal fluids and involved in host defense by pathogen binding by its SRCR domains. Genetic polymorphism within DMBT1 leads to DMBT1-alleles giving rise to polypeptides with interindividually different numbers of SRCR domains, ranging from 8 SRCR domains (encoded by 6 kb DMBT1 variant) to 13 SRCR domains (encoded by the 8 kb DMBT1 variant). In the present study, we have investigated whether reduction from 13 to 8 amino-terminal SRCR domains leads to reduction of bacterial binding. The 6 kb variant bound ~20-45% less bacteria compared to the 8 kb variant. These results support the hypothesis that genetic variation in DMBT1 may influence microbial defense.

Keywords: Genetic polymorphism; Microbial defense; Pathogen binding; SRCR domain.

Fig. 1

DMBT1 polymorphism leads to different length DMBT1 polypeptides. a Schematical presentation of the exon-intron structure within the relevant region of DMBT1 with resulting RsaI restriction fragment sizes depicted below. Gray boxes denote restriction fragments hybridizing with the probe DMBT1/sr1sid2. SR exons coding for scavenger receptor cysteine-rich domains. The hypothetical configurations within the proteins are depicted below. In the carboxy-terminal part of the protein resulting from the deleted allele, it cannot be discerned between a loss of either SR9, SR10, or SR11. Only one of the possibilities is shown. Pink triangle signal peptide, blue box motif without homology, SRCR scavenger receptor cysteine-rich domain, CUB C1r/C1s-Uegf-Bmp1 domains, ZP zona pellucida domain. EHD Ebnerin-homologous domain, orange ovals SRCR interspersed domains (SIDs), TTT and STP are threonine and serine-threonine-proline-rich domains, respectively. b Top panel Southern blot analysis of the DMBT1 genomic configuration in four individuals (A–D) selected from the panel. Band sizes and exons locating on the restriction fragments are depicted at the left. Bottom panel Western blot analysis of DMBT1^SAG protein sizes in the partially purified and concentration-adjusted saliva samples of the four probands. The arrowhead denotes the position of the 220-kDa marker band. DMBT1^SAG was collected from saliva donors that were homozygous for DMBT1/8 kb (donors A and C), homozygous for DMBT1/6 kb (donors B and D). Crude DMBT1^SAG from the four donors samples were separated on 7.5% polyacrylamide gels, transferred to nitrocellulose and immunoblotted with mAb DMBT1H12 . Lane 1 donor A, lane 2, donor B; lane 3, donor C; lane 4, donor D. DMBT1^SAG of donors A and C migrated at a position corresponding to an apparent molecular mass of approximately 340 kDa. DMBT1^SAG of donors B and D runs at a position corresponding to approximately 255 kDa

Fig. 2

Bacterial binding is dependent on DMBT1 polymorphism. a Domain structure of the DMBT1-variant expressed from the large DMBT1 allele (DMBT1/8 kb, 13 SRCR domains within the SRCR/SID region) and the small DMBT1 allele (DMBT1/6 kb, 8 SRCR domains within the SRCR/SID region). Pink triangle leader peptide, blue box sequence contains unique epitope for mAb DMBT1H12, red ovals SRCR domains, orange ovals, SRCR interspersed domains (SIDs), purple boxes C1r/C1s-Uegf-Bmp1 domains, green oval zona pellucida domain, EHD Ebnerin-Homologous Domain. b Bacterial binding to DMBT1/8 kb and DMBT1/6 kb (A) was semi-quantified using S. mutans (S.m), S. gordonii (S.g.), E. coli (E.c.), and H. pylori (H.p.). Relative to the wild type DMBT1^SAG/8 kb we found, on a molecular base, a decrease in bacterial binding to DMBT1^SAG/6 kb for all bacteria tested. Error bars represent the standard error of the mean (SEM), P ≤ 0.05