Glycosylation of dentin matrix protein 1 is a novel key element for astrocyte maturation and BBB integrity
The blood-brain barrier (BBB) is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein 1 (DMP1)-proteoglycan (PG), which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMP1-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 (S89) in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine (S89G), which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that regulated BBB formation, but also assigned a new function to DMP1-PG.DMP1 is an extracellular matrix (ECM) protein. The expression of DMP1 was detected in mouse brain, especially in the blood-brain barrier unit (Fig. 1A). BBB is composed of GFAP positive astrocyte end feet wrapping around blood vessels, which completely seals blood vessels, insulating brain tissues from circulation. DMP1 was found to co-localize with GFAP rather than lectin in BBB units, even though both GFAP and DMP1 were expressed around lectin, which outlined the vasculature. This observation suggested that DMP1 was more associated with astrocytes than endothelial cells.DMP1 can be detected in four protein forms: 57 kDa DMP1 C-terminal (C-DMP1), 37 kDa DMP1 N-terminal (N-DMP1), glycosylated N-terminal core protein (N-DMP1-PG, also referred to as DMP1-PG), and a small amount of non-processed full-length DMP1 (Fig. 1B). A high molecular weight of N-DMP1 was detected by Western blot of brain lysate using N-terminal specific antibodies, which could be converted to 37 kDa core N-DMP1 by Chondroitinase ABC (chABC) (Fig. 1C). To reveal the role of glycosylation of DMP1, we generated a point mutation of DMP1 glycosylation site in a mouse model (S89G-DMP1) through homologous recombination (Fig. 1D). In this model, the glycosaminoglycan (GAG) chain of DMP1 was largely removed (Fig. 1E).S89G-DMP1 knock-in point mutation mouse model was created with homologous recombination method by Beijing Biocytogen Co., Ltd, China. In brief, the S89G mutation was introduced in exon 6 using an overlap extension-PCR method. Homology regions covering 6.6 kb upstream of Dmp1 exon 6 and 8.2 kb downstream of exon 6 were subcloned and FRT-flanked Neo resistance positive selection cassette was inserted into 179 bp downstream of exon 6. The targeting vector was transfected into C57BL/6J embryonic stem (ES) cells by electroporation. Eight positive clones were identified by Southern blotting with 5′ probe and 3′ probe. Three positive clones were injected into Balb/c blastocysts and implanted into pseudopregnant females. Five chimeric male mice were crossed with C57BL/6J females to obtain F1 mice carrying the recombined allele containing the S89G mutation and Neo selection cassette. The presence of the S89G mutation was further verified by sequencing. Heterozygous males were mated with B6.129S4-Gt (ROSA) 26-Sortm1 (FLP1) Dym/RainJ females (Jackson Laboratories), to remove the NEO cassette. Homozygous mutant mice were obtained by intercrossing the heterozygous littermates. Eight positive clones were selected, 3 pups were collected in first litter. Genotyping of S89G-DMP1 mouse primers used for S89G point mutation identification: Dmp1-Neo-F-primer, CGCATTGTCTGAGTAGGTGTC; R-primer, GGTTCTTACATGGGCAGGATAAGC. Transgene PCR product size: 306 bp.DMP1-Tg mouse model was created with Gateway, a bacteriophage-based homologous recombination method by Cyagen Bioscience Inc. (Guangzhou, China). Briefly, we microinjected pRP.ExBi-nestin-Dmp1-IRES-eGFP DNA into mouse zygote to overexpress Dmp1 with nestin promoter. Homozygous transgenic mice were obtained by inter-crossing heterozygous littermates with C57BL/6 background. Two male pups and 4 female pups were collected in first litter. Genotyping of DMP1 mouse-primers used for DMP1-Tg mouse identification: transgene PCR primer F1, AACTTTCCCCGGAGCATCCAC; transgene PCR primer R1, TCTGTACTGGCCTCTGTCGTA; internal control PCR primer F, CAACCACTTACAAGAGACCCGTA; internal control PCR primer R, GAGCCCTTAGAAATAACGTTCACC. Transgene PCR product size: 351 bp; internal control PCR product size: 632 bp.Wild-type C57BL/6 mice were used as control. Pregnant mice were obtained following overnight mating (Day of vaginal plug was defined as embryonic day 0.5). All animals were raised in a specific pathogen-free (SPF) facility, under a 12 h:12 h day–night illumination cycle. For sacrificing mice, animals were killed by cervical dislocation after routine anesthesia. The animal care and use procedures were approved by the Animal Welfare Committee of School of Stomatology, Tongji University (Shanghai, China).Below is the link to the electronic supplementary material.We thank Dr. Chunlin Qin (Bayler colleage of dentistry, Texas A&M University) for providing us DMP1 antibody. This study was supported by China National Key Research and Development Program (2016YFA0100801 YS), and the National Natural Science Foundation of China (Grant Nos. 8133030 YS and 31620103904 YS), and grants: 2016YFC102705 YS; 2014BAI04B07 WZL; 81470715 YS; TJ1504219036 WZL; 2017BR009 YS.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-017-0449-8) contains supplementary material, which is available to authorized users.