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ARYLSULFATASE-I: A NOVEL SULFATASE REGULATING PROTEOGLYCAN SULFATION DURING ENDOCHONDRAL OSSIFICATION

Date

2023-08-01

Journal Title

Journal ISSN

Volume Title

Publisher

ORCID

0000-0002-8010-5931

Type

Thesis

Degree Level

Doctoral

Abstract

Endochondral ossification consists of bone formation from a previous cartilaginous template. This process starts during embryonic development when mesenchymal cells condense in presumptive skeletal regions and differentiate into chondrocytes, which undergo a progressive maturation process. Chondrocytes are arranged in morphologically distinct zones within the cartilaginous template, reflecting their maturation states. While mature chondrocytes are in the medial region, immature chondrocytes are in the distal areas of the template. The chondrocytes pronounced cellular changes during maturation are accompanied by prominent extracellular matrix (ECM) remodelling and reorganization. Sulfated proteoglycans (PGs) are one of the main components of cartilage ECM, and their sulfation levels are critical for proper endochondral ossification. PG sulfation is regulated by sulfotransferases and sulfatases - that add and remove, respectively, sulfate esters from PGs. Deficiencies in sulfatases lead to diseases, including mucopolysaccharidosis and multiple sulfatase deficiency. Disease phenotypes include various skeletal defects seemingly related to chondrocyte maturation, including impaired chondrocyte proliferation and hypertrophy, severe growth retardation, and delayed primary and secondary ossification center formation. The mechanisms of how sulfation leads to those phenotypes and how sulfur levels are modulated across endochondral ossification are still to be unveiled. Here the hypothesis that a novel sulfatase - Arylsulfatase I (ARSI) - decreases PG sulfation during cartilage maturation thus promoting endochondral ossification, was tested. Synchrotron X-ray fluorescence (XRF) imaging confirmed that sulfate esters decreased significantly in mature cartilage during endochondral ossification. ARSB and GALNS- the two known chondroitin sulfate PG sulfatases- were not specifically expressed in mature cartilage. Utilizing laser-capture microdissection and RNAseq data previously obtained in the lab, we were able to identify a novel sulfatase - Arsi - to be upregulated in mature cartilage. Increased ARSI protein expression in mature cartilage was further demonstrated in vivo in chick and mouse sections and during the maturation of ATDC5 chondrocytes in vitro. Biochemical assays overexpressing ARSI in HeLa cells confirmed that ARSI is a novel PG sulfatase. Taken together, biochemical and expression results strongly indicate that ARSI controls the decrease of sulfate esters in mature cartilage. To test ARSI function during endochondral ossification, four putative transgenic zebrafish (Tg) lines for this gene were generated by Gateway-Tol2 transgenesis. All generated Gateway constructs had the correct sequence and were integrated into the zebrafish genome, but possibly due to posttranscriptional mechanisms, protein overexpression was not confirmed in putative Tg fish. Our hypothesis was partially addressed, and more studies are needed to investigate the potential ARSI roles in endochondral ossification. In this thesis, we showed for the first time ARSI mRNA expression in chick, ARSI protein expression in chick and mouse tissues, and we presented the first biochemical characterization of ARSI as a PG sulfatase. Our results supplement the scarce literature about ARSI and potentiate future work uncovering its role in skeletal development and disease.

Description

Keywords

sulfatases, endochondral ossification, cartilage maturation, extracellular matrix

Citation

Degree

Doctor of Philosophy (Ph.D.)

Department

Anatomy, Physiology, and Pharmacology

Program

Anatomy, Physiology, and Pharmacology

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DOI

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