Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is a rare X-linked recessive disease caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). This deficiency leads to a progressive intracellular accumulation of the glycosaminoglycans heparan sulfate and dermatan sulfate, causing severe damage to the central nervous system and other tissues. Previously, we identified a total of 187 IDS-protein interactions in the mouse brain. To validate a subset of these interactions, we selected and cloned the coding region of 10 candidate genes to perform a targeted yeast two-hybrid assay. The results allowed the identification of the physical interaction of IDS with LSAMP and SYT1. Although the physiological relevance of these complexes is unknown, recent advances allow us to point out that these interactions could be involved in vesicular trafficking of IDS through the interaction with SYT1, as well as to the ability to form a transcytosis module between the cellular components of the blood-brain-barrier (BBB) through its interaction with LSAMP. These results may shed light on the role of IDS on cellular homeostasis and may also contribute to the understanding of MPS II physiopathology and the development of novel therapeutic strategies to transport recombinant IDS through the brain endothelial cells toward the brain parenchyma.Funding Information: The project was supported by Pontificia Universidad Javeriana (Project ID 6699), Universidad de Ciencias Aplicadas y Ambientales U.D.C.A (Project ID 15402038 ), the Ministry of Science, Technology and Innovation (Grant No. FP44842-233-2015). EBF received a master’s scholarship from Faculty of Science of Pontificia Universidad Javeriana. CJAD was also supported by Pontificia Universidad Javeriana (Grant ID 3964, 5537, and 7204) and COLCIENCIAS (Grant ID 5174, contract No. 120356933205; and Grant ID 5170, contract No. 120356933427)Declaration of Interests: None to declare. Ethics Approval Statement: Mice were obtained from the Comparative Biology Unit at Pontificia Universidad Javeriana under approved protocols of the Institutional Animal Care and Use Committee. The project approved by the Research and Ethics Committee of the Faculty of Science (PPTA #6699) and by the Institutional Animal Care and Use Committee of Pontificia Universidad Javeriana (FUA #023-15).