Investigation of acidic free-glycans in urine and their alteration in cancer

Abstract

Alterations to glycans in cancer patients have been used to identify novel tumor biomarkers. Most of these studies have focused on protein glycosylation but less attention has been paid to free-glycans. Here, we analyzed acidic free-glycans in the urine of cancer patients to identify novel tumor marker candidates. Specifically, urine samples were collected from patients with gastric cancer, pancreatic cancer and cholangiocarcinoma as well as normal controls. The free-glycans were extracted from creatinine-adjusted urine and fluorescently labeled with 2-aminopyridine. Initially, we performed profiling of urinary free-glycans by high-performance liquid chromatography and mass spectrometry with enzymatic and chemical degradation. More than 100 glycans, including novel structures, were identified. The chromatographic peaks suggested some of these glycans were present at elevated levels in cancer patients. To verify cancer-associated alterations, we compared the glycan levels between cancer patients and normal controls by selected reaction monitoring. Representative structures of glycans with elevated levels in cancer patients included the following: small glycans related to sialyllactose; sialyl Lewis X; lactose- and N-acetyllactosamine (LacNAc) type-II-core glycans with LacNAc (type-I or II)-extensions and modifications of α1,3/4-fucose and/or 6-sulfate on the Glc/GlcNAc; free-N-glycans containing sialylation or β1,6-branch of 6-sulfo Lewis X; novel NeuAcα2-3Galβ1–4(+/−Fucα1–3)Xylα1–3Glc glycans. Our results provide further insight into urinary free-glycans and suggest the potential utility of these compounds as tumor markers.

Occurrence of a d-arabinose-containing complex-type free-N-glycan in the urine of cancer patients

“…Clinical information on all specimens is shown in Supplementary Table S1 . Urinary free-glycans were prepared as previously reported for acidic glycans 16 . The reducing ends of the free-glycans purified from creatinine-corrected urine were labeled with a fluorophore, 2-aminopyridine (PA), by a reductive amination reaction.…”

“…Our previous study focusing on acidic free-glycans identified various candidate tumor markers that are elevated in cancer patients, including novel glycan structures 16 . These findings prompted us to search for tumor markers of neutral free-glycans in urine.…”

“…Urinary free-glycans were prepared as previously reported 16 . Briefly, the volume of the urine specimens used for sample preparation were corrected to normalize for creatinine concentration.…”

“…Our initial studies focused on identifying tumor markers comprising acidic free-glycans. The structures of 135 free-glycans were identified, and several free-glycans that were elevated in the urine of gastric, pancreatic cancer and cholangiocarcinoma patients found 16 . In a subsequent study, structural analyses of neutral free-glycan in urine of healthy subjects were conducted 17 .…”

Direct N-Glycosylation Profiling of Urine and Prostatic Fluid Glycoproteins and Extracellular Vesicles

“…One of the most highly characterized glycoproteins is PSA (White et al, 2009;Leymarie et al, 2013;Nyalwidhe et al, 2013;Jia et al, 2017;Kammeijer et al, 2018;Hatakeyama et al, 2021), due to its known role in prostate cancer diagnosis and relatively simple glycosylation pattern of having a single N-linked glycosylation site. Although changes in glycosylation of PSA and many other prostatic glycoproteins have diagnostic potential, assays to efficiently characterize N-glycans in urine can be lengthy and require multiple processing steps, precluding large scale clinical utility (Song et al, 2019;Hanzawa et al, 2021). Lectin arrays have been effectively used to profile glycan motifs in large cohorts of clinical urine samples (Kawakita et al, 2021;Mise et al, 2021), however this approach cannot determine full glycan compositions or distinguish N-linked or O-linked origins.…”