# Expression of Heart Development Protein With EGF‐Like Domains 1 (HEG1) Decorated With Low‐Sulfated Keratan Sulfate in Human Malignant Pleural Mesothelioma

## TL;DR

This study shows that a specific protein, HEG1, decorated with low-sulfated keratan sulfate, is present in malignant pleural mesothelioma and could be a new diagnostic or therapeutic target.

## Contribution

The study identifies HEG1 as a core protein for low-sulfated keratan sulfate in malignant pleural mesothelioma, offering new insights into diagnostic markers and potential therapies.

## Key findings

- Most MPM tissues express low-sulfated keratan sulfate detectable by specific monoclonal antibodies.
- HEG1 is confirmed as a core protein for low-sulfated keratan sulfate in MPM.
- Low-sulfated keratan sulfate glycoforms on HEG1 may serve as a new therapeutic target for MPM.

## Abstract

The glycoform of heart development protein with EGF‐like domains 1 (HEG1) recognized by the SKM9‐2 monoclonal antibody is a useful diagnostic marker for malignant pleural mesothelioma (MPM). The putative glycoform includes core 2 O‐glycans carrying sialyl poly‐N‐acetyllactosamine (LacNAc), but sulfation modifications are undetermined. Since sialyl 6‐sulfo LacNAc‐capped core 2 O‐glycans are expressed in MPM and their structure overlaps with low‐sulfated keratan sulfate (KS), we asked whether low‐sulfated KS is expressed in MPM and whether HEG1 is decorated with low‐sulfated KS. We performed immunohistochemical analysis of 41 MPM cases using anti‐KS monoclonal antibodies and endoglycosidases, reversed‐phase ion‐pair high‐performance liquid chromatography analysis of KS/sulfated LacNAc isolated from human pleural tissue, and western blot analysis of HEG1·IgG recombinant fusion proteins secreted from low‐sulfated KS‐expressing human embryonic kidney cells. Most MPM tissues were stained with anti‐low‐sulfated KS antibodies and staining was eliminated by endo‐β‐galactosidase and keratanase II but not by peptide‐N‐glycosidase F. Disaccharide composition analysis revealed that mono‐sulfated LacNAc disaccharide and di‐sulfated LacNAc disaccharide accounted for 83.1% and 16.9% of pleural KS/sulfated LacNAc, respectively. Western blot analysis of HEG1·IgG glycoforms indicated that HEG1 functions as a core protein for low‐sulfated KS. Thus, HEG1 protein decorated with low‐sulfated KS is expressed in MPM.

The purpose of this study is to investigate sulfation modification of the glycan structure decorating HEG1, one of the diagnostic markers of malignant pleural mesothelioma (MPM), in MPM. Immunohistochemical analysis reveals low‐sulfated keratan sulfate (KS) expression, recognized by 294‐1B1 and R‐10G, in MPM tissues with HEG1 expression. Western blot analysis demonstrates that HEG1 functions as a core protein for low‐sulfated KS, suggesting the glycoform may be a potential target for new therapy for MPM.