Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1

Our plain-English summary of Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1, by R. Matsuura, H. Kaji, A. Tomioka, et al (Scientific Reports, 2018).

The study

• Authors: R. Matsuura, H. Kaji, A. Tomioka, T. Sato, H. Narimatsu, Y. Moriwaki, H. Misawa, K. Imai, S. Tsuji
• Journal: Scientific Reports
• Published: 2018;8(1):14251
• Cited by: 16 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The anti-mesothelioma mAb SKM9-2 recognizes the sialylated protein HEG homolog 1 (HEG1). HEG1 is a 400 kDa mucin-like membrane protein found on mesothelioma. SKM9-2 can detect mesothelioma more specifically and sensitively than other antibodies against current mesothelioma markers; therefore, SKM9-2 would be likely useful for the precise detection and diagnosis of malignant mesothelioma. In the present study, we investigated the epitope of SKM9-2. We analyzed the binding of SKM9-2 to truncated HEG1 and candidate epitope-fused glycosylphosphatidylinositol-anchor proteins. The epitope of SKM9-2 was identified as an O-glycosylated region, 893-SKSPSLVSLPT-903, in HEG1. An alanine scanning assay of the epitope showed that SKM9-2 bound to a simple epitope in HEG1, and the SKxPSxVS sequence within the epitope was essential for SKM9-2 recognition. Mass spectrometry analysis and lectin binding analysis of soluble epitope peptides indicated that the SKM9-2 epitope, in which Ser897 was not glycosylated, contained two disialylated core 1 O-linked glycan-modified serine residues, Ser893 and Ser900. Neuraminidase treatment analysis also confirmed that the epitope in mesothelioma cells contained a similar glycan modification. The specific detection of mesothelioma with SKM9-2 can thus be performed by the recognition of sialylated glycan modification in the specific region of HEG1.

In plain English

This laboratory study examined where an antibody called SKM9-2 attaches to HEG1, a sugar-coated protein on mesothelioma cells. Using human cell lines, including two mesothelioma lines, the authors tested which shortened or altered versions of HEG1 the antibody recognized. No asbestos exposure was studied. SKM9-2 bound HEG1 positions 893 to 903, and changing any of several building blocks there stopped binding. The antibody needed sugar chains at positions 893 and 900 and did not bind the stretch without sugars. The main form it bound carried a sugar type called disialyl T there. However, the authors reported that it was not strict about the exact pattern of sialic acid, a sugar unit. With a purified lab-made copy of that stretch, it still bound briefly after all sialic acid was removed. On mesothelioma cells, removing all sialic acid ended binding. The authors concluded that this recognition could be useful for detecting and diagnosing malignant mesothelioma. They noted that they could not directly identify the sugars on natural HEG1 from mesothelioma cells, and that sugar variations in real tissue might affect detection.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (cell-line and protein chemistry) study mapping the binding site (epitope) of the monoclonal antibody SKM9-2 on the protein HEG1
• Who was studied: No patients. The authors used the human malignant mesothelioma cell lines ACC-MESO-1 and ACC-MESO-4, the human embryonic kidney cell line HEK293T, and 293 H cells, which they used to produce HEG1 and HEG1 fragments.
• Exposure: No asbestos or fiber exposure was studied. Asbestos appears only in the introduction's background statement about the cause of mesothelioma.
• Main results:
  • Tests with shortened HEG1 fragments located the SKM9-2 binding region at positions 893 to 903 of HEG1 (sequence SKSPSLVSLPT).
  • Replacing any one of six positions in this region (Ser893, Lys894, Pro896, Ser897, Val899 or Ser900) with alanine stopped SKM9-2 from recognizing it.
  • SKM9-2 binding required sugar chains (O-linked glycans) on Ser893 and Ser900, with Ser897 left without sugar. The antibody did not bind the peptide without sugars.
  • The main, strongly binding form of the epitope carried two disialyl T sugar chains, at Ser893 and Ser900.
  • The antibody was not strict about the number or linkage of sialic acids.
  • With the purified, lab-made short epitope peptide (SKMepmin3), SKM9-2 could still interact after the sialic acids were removed, although it came off quickly (the dissociation rate was high).
  • In the mesothelioma cell lines ACC-MESO1 and ACC-MESO4, removing sialic acids with Neuraminidase A completely abolished SKM9-2 binding to the epitope-carrying protein.
  • In natural HEG1 from the mesothelioma cell line ACC-MESO4, SKM9-2 binding was weakened by one sialic-acid-removing enzyme (α2-3 Neuraminidase S) and lost with another (Neuraminidase A). The authors took these results to suggest that the epitope on mesothelioma cells carries disialyl T in the same way as the lab-made peptide.
• The authors' conclusion: The authors concluded that SKM9-2's recognition of sugar modification in this specific region of HEG1 could be useful for precise detection and diagnosis of malignant mesothelioma.
• Limitations the authors state:
  • The authors could not directly identify the sugar chains in the binding site of natural HEG1 on mesothelioma cells, because they could not obtain the digested epitope peptides.
  • The peptides were produced by cell lines grown in serum-free media. The authors noted that sialic acid variants in real tumor tissue may affect the sensitivity and specificity of mesothelioma detection, and that further analysis may be needed.
  • Large sugar chains near the binding site might interfere with SKM9-2 binding. The authors stated that this should be investigated in different histological subtypes of mesothelioma.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelioma, Monoclonal Antibody, HEG1.

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• Tissue distribution of a coelomic-epithelium-related antigen recognized by the monoclonal antibody OC125 — S.E. Kabawat, R.C. Bast, et al. (1983)
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