N‐glycosylation status of β‐haptoglobin in sera of patients with colon cancer, chronic inflammatory diseases and normal subjects

Abstract

N‐glycosylation status of purified β‐haptoglobin from sera of 17 patients, and from sera of 14 healthy volunteer subjects, was compared by blotting with various lectins and antibodies. Patients in this study were diagnosed as having colon cancer through histological examination of each tumor tissue by biopsy. Blotting index of serum β‐haptoglobin with Aleuria aurantia lectin (AAL) was clearly higher for cancer patients than for healthy subjects. No such distinction was observed for blotting with three other lectins and two monoclonal antibodies. To determine tumor‐associated reactivity of AAL binding as compared to inflammatory processes in colonic tissues, β‐haptoglobin separated from sera of 5 patients with Crohn's disease (CD), and 4 patients with ulcerative colitis (UC), was studied. All these cases, except one case of UC, showed AAL index lower than that in cancer cases, similarly to healthy subjects. The higher AAL binding of β‐haptoglobin in colon cancer patients than in healthy subjects appeared to be due to α‐L‐fucosyl residue, since it was eliminated by bovine kidney α‐fucosidase treatment. N‐linked glycans of serum haptoglobin from colon cancer patients vs. healthy subjects were released by N‐glycanase, fluorescence‐labeled, and subjected to normal‐phase high performance liquid chromatography (NP‐HPLC). Glycan structures were determined based on glucose unit (GU) values and their changes upon sequential treatment with various exoglycosidases. Glycosyl sequences and their branching status of glycans from 14 cases of serum β‐haptoglobin were characterized. The identified glycans were sialylated or nonsialylated, bi‐antennary or tri‐antennary structures, with or without terminal fucosylation.

Dimeric Lea (Lea-on-Lea) status of β-haptoglobin in sera of colon cancer, chronic inflammatory disease and normal subjects

“…This result is similar to that of our previous study on blotting of Hap with Aleuria aurantica lectin (AAL), which also shows higher reactivity with colorectal cancer (28). Both Le a -on-Le a structure, and the unknown structure defined by AAL, contain ·-L-fucosyl residue.…”

“…For defucosylation, 5 μg of Hap was incubated with/without 1 mU bovine kidney ·-L-fucosidase in 50 mM sodium acetate buffer (pH 5.5) for 24 h at 37˚C as described previously (28). The reaction of de-N-glycosylation was performed by incubation of 5 μg Hap with/without 15 mU PNGase F according to the manufacturer's instructions.…”

“…Purification of Hap was performed as described previously (28). Briefly, each serum was diluted 4 times with PBS, subjected into rabbit anti-Hap antibodyconjugated sepharose 4B equilibrated with PBS, and incubated for 2 h at room temperature by shaking.…”

Site-specific Glycoforms of Haptoglobin in Liver Cirrhosis and Hepatocellular Carcinoma

“…Lin et al (42) reported increased core fucosylation in patients with pancreatic cancer; both core and outer arm fucoses were detected among the doubly fucosylated glycans. Dimeric Lewis-A type (Le(a) on Le(a)) structures of Hp were found increased in colon cancer (43). Bi-antennary N-acetyllactosamine glycans with increased binding to galectin-1 have been detected on Hp in metastatic breast cancer (6).…”

Identification of proteins with the CDw75 epitope in human colorectal cancer

“…However, Hp cannot be the only protein that bears this epitope as there are heavier bands present in immunoblots of CDw75 compared with immunoblots of Hp. In previous studies, the sequence Neu5Ac-Gal-GlcNAc has been detected among the multiple carbohydrate determinants that are attached to serum Hp (45,46). Given the complex histological composition of the CRC specimens employed in the present study, and the fact that it was not possible to detect any signal of the protein in Caco-2 and HT-29 cell lines by WB, the exact origin of the Hp detected by MS cannot be elucidated, although, as stated previously the protein is known to be expressed in the colorectal epithelium (42,43).…”