Collagen is the most abundant protein in the extracellular matrix of animals, and 28 types of collagen have been reported in humans. We previously analyzed the endoplasmic reticulum (ER)-to-Golgi transport of fibril-forming type III collagen (Hirata et al. 2022) and network-forming type IV collagen (Matsui et al. 2020), both of which have long collagenous triple-helical regions. To understand the ER-to-Golgi trafficking of various types of collagens, we analyzed the transport of short-chain type X collagen in this study. We fused cysteine-free GFP to the N-telopeptide region of procollagen X (GFP-COL10A1), as employed in our previous analysis of procollagens III and IV, and analyzed its transport by live-cell imaging. Procollagen X was transported to the Golgi apparatus via vesicular and tubular carriers containing ERGIC53 and RAB1B, similar to those used for procollagen III. Carriers containing procollagen X probably used the same transport processes as those containing conventional cargoes such as ⍺1-antitrypsin. SAR1, TANGO1, SLY1/SCFD1, and BET3/TRAPPC3 were required for trafficking of procollagen X, which are different from the factors required for trafficking of procollagens III (SAR1, TANGO1, and CUL3) and IV (SAR1 and SLY1/SCFD1). These findings reveal that accommodation of various types of collagens with different shapes into carriers may require fine-tuning of the ER-to-Golgi transport machinery.Key words: collagen, GFP-procollagen X, ER-to-Golgi trafficking, export from ER, TANGO1.

The obligatory intracellular bacterium Anaplasma phagocytophilum causes human granulocytic anaplasmosis, an emerging zoonosis. Anaplasma has limited biosynthetic and metabolic capacities, yet it effectively replicates inside of inclusions/vacuoles of eukaryotic host cells. Here, we describe a unique Type IV secretion system (T4SS) effector, ER-Golgi exit site protein of Anaplasma (EgeA). In cells infected by Anaplasma, secreted native EgeA, EgeA-GFP, and the C-terminal half of EgeA (EgeA-C)-GFP localized to Anaplasma-containing inclusions. In uninfected cells, EgeA-C-GFP localized to cis-Golgi, whereas the N-terminal half of EgeA-GFP localized to the ER. Pull-down assays identified EgeA-GFP binding to a transmembrane protein in the ER, Transport and Golgi organization protein 1 (TANGO1). By yeast two-hybrid analysis, EgeA-C directly bound Sec1 family domain-containing protein 1 (SCFD1), a host protein of the cis-Golgi network that binds TANGO1 at ER-Golgi exit sites (ERES). Both TANGO1 and SCFD1 localized to the Anaplasma inclusion surface. Furthermore, knockdown of Anaplasma EgeA or either host TANGO1 or SCFD1 significantly reduced Anaplasma infection. TANGO1 and SCFD1 prevent ER congestion and stress by facilitating transport of bulky or unfolded proteins at ERES. A bulky cargo collagen and the ER-resident chaperon BiP were transported into Anaplasma inclusions, and several ER stress marker genes were not up-regulated in Anaplasma-infected cells. Furthermore, EgeA transfection reduced collagen overexpression-induced BiP upregulation. These results suggest that by binding to the two ERES proteins, EgeA redirects the cargo-adapted ERES to pathogen-occupied inclusions and reduces ERES congestion, which facilitates Anaplasma nutrient acquisition and reduces ER stress for Anaplasma survival and proliferation.

TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion.

The endoplasmic reticulum (ER) to Golgi secretory pathway is an elegantly complex process whereby protein cargoes are manufactured, folded, and distributed from the ER to the cisternal layers of the Golgi stack before they are delivered to their final destinations. The export of large bulky cargoes such as procollagen and its trafficking to the Golgi is a sophisticated mechanism requiring TANGO1 (Transport ANd Golgi Organization protein 1. It is also called MIA3 (Melanoma Inhibitory Activity protein 3). TANGO1 has two prominent isoforms, TANGO1-Long and TANGO1-Short, and each isoform has specific functions. On the luminal side, TANGO1-Long has an HSP47 recruitment domain and uses this protein to collect collagen. It can also tether its paralog isoforms cTAGE5 and TALI and along with these proteins enlarges the vesicle to accommodate procollagen. Recent studies show that TANGO1-Long combines retrograde membrane flow with anterograde cargo transport. This complex mechanism is highly activated in fibrosis and promotes the excessive deposition of collagen in the tissues. The therapeutic targeting of TANGO1 may prove successful in the control of fibrotic disorders. This review focuses on TANGO1 and its complex interaction with other procollagen export factors that modulate increased vesicle size to accommodate the export of procollagen.

Newly synthesized secretory proteins are exported from the endoplasmic reticulum (ER) at specialized subcompartments called exit sites (ERES). Cargoes like procollagen are too large for export by the standard COPII-coated vesicle of 60 nm average diameter. We have previously suggested that procollagen is transported from the ER to the next secretory organelle, the ER-Golgi intermediate compartment (ERGIC), in TANGO1-dependent interorganelle tunnels. In the theoretical model presented here, we suggest that intrinsically disordered domains of TANGO1 in the ER lumen induce an entropic contraction, which exerts a force that draws procollagen toward the ERES. Within this framework, molecular gradients of pH and/or HSP47 between the ER and ERGIC create a force in the order of tens of femto-Newtons. This force is substantial enough to propel procollagen from the ER at a speed of approximately 1 nm · s-1. This calculated speed and the quantities of collagen secreted are similar to its observed physiological secretion rate in fibroblasts, consistent with the proposal that ER export is the rate-limiting step for procollagen secretion. Hence, the mechanism we propose is theoretically adequate to explain how cells can utilize molecular gradients and export procollagens at a rate commensurate with physiological needs.

Dental-skeletal-retinal-anomaly (DSRA) is a newly described collagenopathy in Cane Corso dogs. The causative mutation has been linked with splice defects within the melanoma inhibitory activity member 3 (MIA/3) gene that codes for the TANGO1 protein. This case series presents the first dental-related radiographic and histopathological abnormalities in two dogs with genetically confirmed DSRA. The clinical, radiological, and histological features are similar to those reported for MIA3/TANGO1 splice defects previously reported in humans and knockout mice. Common clinical features of these patients include generalized opalescent discoloration of the permanent dentition (intrinsic dyschromia), enamel defects, fractured teeth, vision loss, shortened physical stature, and orthopedic abnormalities that resulted in chronic, early-onset lameness. Intraoral radiography revealed delayed dentin deposition, evidence of endodontic disease, and dental hard tissue loss in both cases. Histopathologic findings for both cases were consistent with dentinogenesis imperfecta (DGI). DSRA exhibits autosomal recessive heritability and commercial diagnostic tests are now available. Clinicians should be aware of the etiopathogenesis, genetic inheritance and associated comorbidities in order to treat and counsel clients on the management of this condition. It is recommended that all breeding individuals be tested, and carriers be sterilized or omitted from the breeding population. This case study describes intraoral diagnoses, treatments, and follow-up of two DSRA-positive dogs.

Transport and Golgi organization 1 (TANGO1) also known as MIA3, belongs to the melanoma inhibitory activity gene (MIA) family together with MIA, MIA2 and OTOR; these members play different roles in different tumors, but the mechanism underlying TANGO1s effect on hepatocellular carcinoma (HCC) is unclear. Our study confirmed that TANGO1 is a promoter of HCC, In HCC cells, TANGO1 can promote proliferation, inhibit apoptosis, promote EMT. These changes were reversed after TANGO1 inhibition. We explored the molecular mechanism of TANGO1 and HCC and found that the promoting effect of TANGO1 on HCC related to neurturin (NRTN) and the PI3K/AKT/mTOR signaling pathway based on RNA-seq results. NRTN is not only related to neuronal growth, differentiation and maintenance but is also involved in a variety of tumorigenic processes, and PI3K/AKT/mTOR signaling pathway has been shown to be involved in HCC progression. We verified that TANGO1 interacts with NRTN in HCC cells using endogenous Co-IP and confocal localization, and both promote HCC progression by activating the PI3K/AKT/mTOR signaling pathway. Our results reveal the mechanism by which TANGO1 promotes HCC progression, suggesting that the TANGO1/NRTN axis may be a potential therapeutic target for HCC worthy of further investigation.

BACKGROUND: Collagen, the most abundant human protein, is a significant component of the extracellular matrix (ECM) in tissues and organs like skin, bone, ligaments, and tendons. Collagen secretion is a complex, multistage process involving many molecules. A protein playing one of the main functions in this process is TANGO1 encoded by MIA3 gene. In the hypermobile type of Ehlers-Danlos syndrome (hEDS), one of the most common collagenopathies with no known genetic background, disrupted secretion of many molecules (including collagen) was observed.
OBJECTIVE: The aim of this study was the evaluation of the MIA3 gene role in hEDS patients.
METHODS: One hundred patients with clinically diagnosed hEDS and negative next-generation sequencing (NGS) testing for connective tissue disorder (e.g. Ehlers-Danlos syndrome, osteogenesis imperfect (OI), Marfan syndrome, and others) were tested for molecular changes in the MIA3 gene.
RESULTS: Among the 100 tested patients, 14 single structural changes in the MIA3 gene were detected. Thirteen were missense benign or likely benign, while 1 variant (c.567dup, p.Leu1880ThrfsTer6) was truncating the TANGO1 protein.
CONCLUSIONS: We suppose that the presence of truncating variant (c.5637dup) in the MIA3 gene and disrupted secretion of connective tissue protein may be one of the pathogenic mechanisms of clinical symptoms present in the tested patient, but these findings require a more comprehensive multidimensional investigation.

Lack of type VII collagen (C7) disrupts cellular proteostasis yet the mechanism remains undescribed. By studying the relationship between C7 and the extracellular matrix (ECM)-associated proteins thrombospondin-1 (TSP1), type XII collagen (C12) and tissue transglutaminase (TGM2) in primary human dermal fibroblasts from multiple donors with or without the genetic disease recessive dystrophic epidermolysis bullosa (RDEB) (n=31), we demonstrate that secretion of each of these proteins is increased in the presence of C7. In dermal fibroblasts isolated from patients with RDEB, where C7 is absent or defective, association with the COPII outer coat protein SEC31 and ultimately secretion of each of these ECM-associated proteins is reduced and intracellular levels are increased. In RDEB fibroblasts, overall collagen secretion (as determined by the levels of hydroxyproline in the media) is unchanged while traffic from the ER to Golgi of TSP1, C12 and TGM2 occurs in a type I collagen (C1) dependent manner. In normal fibroblasts association of TSP1, C12 and TGM2 with the ER exit site transmembrane protein Transport ANd Golgi Organization-1 (TANGO1) as determined by proximity ligation assays, requires C7. In the absence of wild-type C7, or when ECM-associated proteins are overexpressed, C1 proximity and intracellular levels increase resulting in elevated cellular stress responses and elevated TGFβ signaling. Collectively, these data demonstrate a role for C7 in loading COPII vesicle cargo and provides a mechanism for disrupted proteostasis, elevated cellular stress and increased TGFβ signaling in patients with RDEB. Furthermore, our data point to a threshold of cargo loading that can be exceeded with increased protein levels leading to pathological outcomes in otherwise normal cells.

We investigated a hereditary syndrome in Cane Corso dogs. Affected dogs developed dental-skeletal-retinal anomaly (DSRA), clinically characterized by brittle, discolored, translucent teeth, disproportionate growth and progressive retinal degeneration resulting in vision loss. Combined linkage and homozygosity mapping delineated a 5.8 Mb critical interval. The comparison of whole genome sequence data of an affected dog to 789 control genomes revealed a private homozygous splice region variant in the critical interval. It affected the MIA3 gene encoding the MIA SH3 domain ER export factor 3, which has an essential role in the export of collagen and other secreted proteins. The identified variant, XM_005640835.3:c.3822+3_3822+4del, leads to skipping of two exons from the wild type transcript, XM_005640835.3:r.3712_3822del. Genotypes at the variant were consistent with monogenic autosomal recessive mode of inheritance in a complete family and showed perfect genotype-phenotype association in 18 affected and 22 unaffected Cane Corso dogs. MIA3 variants had previously been shown to cause related phenotypes in humans and mice. Our data in dogs together with the existing functional knowledge of MIA3 variants in other mammalian species suggest the MIA3 splice defect and a near complete loss of gene function as causative molecular pathomechanism for the DSRA phenotype in the investigated dogs.