Research progress on O-GlcNAcylation in the occurrence, development, and treatment of colorectal cancer

Table 1 Summary of Studies on O-GlcNAcylation and colorectal cancer in recent 5 years

Ref.	Year	Clinical specimens	Cell source	Methods	Conclusion
Madunić et al[21]	2020	Human CRC cell lines.	(1) Department of Surgery of the LUMC, Leiden, The Netherlands. And (2) Department of Pathology of the VUMC, Amsterdam, The Netherlands	(1) OGlycan release and analysis. And (2) Glycan structure analysis and relative quantification	Further untargeted screening of cell line O-GlcNAcylation paves the way for further exploration of the role of glycosylation in CRC development and drug response, thus identifying new anticancer antibody development targets
Gao et al[93]	2020	LS174T Tn (+), LS174T Tn (-) and LSC cells	Professor Tongzhong Ju, Emory University School of Medicine, Atlanta, United States	(1) Vector construction and cell transfection. (2) Exosome isolation and purification. (3) RNA extraction and qRT-PCR. (4) Protein extraction, deglycosylated preparation, and Western blotting (WB). And (5) Flow cytometry analysis.	CD44 in exosomes may be a potential biomarker for abnormal O-GlcNAcylation. This is the first study to show that abnormal O-GlcNAcylation can affect the expression or delivery of O-glycoproteins through exosomes, providing a new perspective for our study of treatment strategies for human colon cancer
Gao et al[61]	2020	(1) The tumor tissues were freshly acquired by surgical resection. And (2) Normal colorectal mucosa were taken at biopsy from individuals without colorectal malignancies	Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China	(1) RNA extraction and qRT-PCR. (2) WB. (3) Lentivirus-mediated COSMC transfection. (4) Transwell migration and invasion assays. And (5) Flow cytometry analysis	The increased expression of COSMC in human CRC may be caused by endoplasmic reticulum stress, which further enhances malignant tumors by activating EMT without dependence on abnormal O-GlcNAcylation
Kvorjak et al[54]	2020	(1) SW480 (ATCC® CCL-228) and HT-29 (ATCC® HTB-38) cell Lines. And (2) Archived paraffin sections of colonic biopsies and those with colitis associated colon cancer	(1)American Type Culture Collection. And (2) Department of Gastroenterology, University of Pittsburgh, United States	(1) WB. (2) Immunofluorescence confocal microscopy. (3) IHC. (4) Migration and invasion assay. (5) Peripheral blood monocyte isolation and macrophage differentiation. (6) Indirect co-culture assay. Cytokine and chemokine expression detection. (7) Gene expression profiling. Chromatin IP assay. (8) qRT-PCR. And (9) Computational modeling and simulation	To construct a computational model of a signal pathway and detect the inhibitory effect of IL-13 as a possible therapeutic method. Our findings reveal a new cell crossover between colon cells and macrophages in the inflammatory and malignant colon, which contributes to the pathogenesis of colitis-associated CRC
Liu et al[65]	2019	(1) The human CRC cell lines HCT116 and SW480. (2) The human embryonic kidney cells HEK293T. And (3) The human CRC cells LS174T (Tn-positive)	(1) American Type Culture Collection (ATCC). And (2) Dr. Tongzhong Ju of the Emory University School of Medicine, Atlanta, United States	(1) CRISPR/Cas9-mediated knockout of COSMC chaperone. (2) Flow cytometry. (3) Cell migration and invasion assays. (4) Establishment of transplantable metastatic murine models. (5) IHC. (6) Knockdown of H-Ras with shRNA. (7) Re-expression of COSMC in LS174T cells. (8) RNA extraction and qRT-PCR. (9) WB and antibodies. And (10) TCGA colon cancer dataset	Tn antigen expression (a marker of abnormal O-GlcNAcylation) may promote EMT activation by upregulation of h-RAS, possibly leading to CRC metastasis. It also suggests that anti-Tn antigen has a great prospect in tumor immunotherapy
Biwi et al[22]	2019	Human fetal colon CCD841CoN, colon adenocarcinoma HT29, and colon carcinoma HCT116 cells	LUMC, Centre for Proteomics and Metabolomics 2333ZA Leiden, Netherlands	(1) Transcriptomic. (2) IP and WB. (3) Lectin labeling and flow cytometry analysis. (4) Indirect IF and confocal microscopy. (5) Cell harvest for MS. (6) N-glycan release from cell lysates and MS analysis. (7) O-glycan release and MS analysis. And (8) Glycosphingolipid analysis by MS	OGT silencing in HT29 cells upregulates E-cadherin (the main role of epithelial to mesenchymal transition) and changes its glycosylation. Alternatively, OGT silencing interferes with glycophosphatidylcholine biosynthesis, decreasing gangliosides and the increase of globular glycosides. In conclusion, these results provide new insight into the selective regulation of complex glycosylation of O-GlcNAcylation in CRC cells
Zhu et al[27]	2019	(1) The human colon cancer tissue microarray analysis (TMA). (2) Colon cancer tissues and adjacent normal colon tissues. And (3) The CRC cell lines SW480, HCT116, LoVo, COLO205, HT29, CaCo-2 and colonic epithelial cell line NCM-460	(1) Shanghai Tenth People’s Hospital. And (2) Cell Bank of the Chinese Academy of Sciences. Shanghai, China	(1) IHC, IF, and WB. (2) qRT-PCR. (3) Cell proliferation, Caspase 3/7 activity and soft agar colony formation assay. (4) Co-IP. (5) Chromatin IP. (6) Protein ligation assay (PLA). (7) In vitro O-GlcNAcylation of YY1. (8) Enzymatic labelling of O-GlcNAc sites. (9) Mice experiments. And (10) Bioinformatics analysis	The O-GlcNAcylation of YY1 by SLC22A15 and AANAT provoked oncogenesis of CRC cells, indicating that YY1 O-GlcNAcylation might be a potential effective target for the treatment of CRC
Yu et al[48]	2019	CRC tumor tissue	CHINA-JAPAN Union Hospital of Jilin University	(1) IHC. (2) Cell culture and treatment. (3) Lentivirus obtainment and stable cell lines establishment. (4) qRT-PCR and WB. (5) Cell counting Kit-8 (CCK-8) assay. (6) Flow cytometry assay and IP. (7) In vitro O-GlcNAcylation of ITGA5. (8) Enzymatic labelling of shi'yO-GlcNAc sites. And (9) Xenotransplantation	ITGA5 was highly expressed in CRC tissues and cells, and with increased OGlcNAcylation, its stability was higher, thereby promoting cell proliferation and tumor formation, and reducing apoptosis
Jiang et al[52]	2019	(1) CRC tissue microarrays (HCol-Ade180Sur-09). And (2) Primary CRC tissues and paired adjacent normal tissues samples	(1) Shanghai Outdo Biotech. And (2) Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi an, China	(1) Cell culture. (2) Virus packaging. (3) The construction of human full-length OGT (NM_181672). And (4) Dual-luciferase reporter assay	In CRC cells, miR-101/O-GlcNAcylation/ EZH2 signaling forms a feedback loop that promotes metastasis, providing a new insight into the basic theory of tumor metastasis and treatment strategies
Wu et al[93]	2019	Human CRC cell lines HT29, HCT116, SW480, SW620, and normal intestinal epithelial cells NCM460	Chinese Academy of Sciences Cell Bank, China	(1) RNA extraction, reverse transcription, and qRT-PCR. (2) Cycloheximide or Thiamet-G treatment. (3) WB and Co-IP. (4) Cell viability assay. (5) Colony formation assay. (6) ICH and IF. (7) Cell migration assay. (8) mTOR agonist and inhibitor treatment. (9) Lentivirus production and infection. And (10) Tumorgenicity assay in nude mice	By strengthening the stability of RNA helicase P68 (DDX5) and the activation of AKT/mTOR signaling pathway, the elevation of O-GlcNAcylation significantly promoted the proliferation and metastasis of CRC cells, and manifest a poor prognosis
Ubillos et al[72]	2018	(1) Human CRC cell lines HT29 (ATCC HTB38™), SW480 [SW-480] (ATCC CCL228™) and SW620. [SW620] (ATCC CCL227™). (2) Adenomas with different degrees of dysplastic lesions. And (3) Normal colon tissues from distal or proximal resection margin	Department of Pathology, Maciel Hospital, Montevideo	(1) qRT-PCR. (2) IF microscopy. (3) Analysis of GalNAc-T6 expression on cancer cell lines by flow cytometry. And (4) IHC	The molecular mechanism by which GalNAc-T6 expression predicts improved prognosis in CRC patients with reduced invasiveness in CRC cells expressing GalNAc-T6 is unclear
Sun et al[15]	2018	(1) Paraffin-embedded tissue sections. And (2) Frozen tissues	Emory University School of Medicine, Atlanta, United States	(1) IF. (2) Flow cytometry. (3) FACS. (4) IHC. (5) WB. (6) T-synthase activity assay. (7) Genomic DNA preparation. (8) LOH and mutation analyses. And (9) Total RNA extraction and qRT-PCR	The loss of T-synthetase/COSMC due to genetic and epigenetic inactivation of COSMC may be responsible for the expression of Tn in human CRC cell lines and pancreatic cancer. Simultaneously, there are other mechanisms in Tn positive CRC
Jiang et al[14]	2018	(1) The human primary/metastatic CRC and adjacent normal tissues. And (2) The human CRC LS174T cell line (Tn-positive)	(1) Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China. And (2) Provided by Dr. Tongzhong Ju of Emory University School of Medicine in Atlanta, United States	(1) Immunohistochemical staining of Tn antigen. (2) Exome sequencing. (3) Analysis of DNA methylation by MALDI-TOF mass spectrometry. (4) T-synthase activity assay. (5) RNA extraction and qRT-PCR. (6) WB. (7) Lentiviral-mediated COSMC transfection. (8) Flow cytometry analysis. (9) Cell proliferation, migration, and apoptosis. And (10) Multiplex IHC staining	The expression of MUC2, which plays an essential role in intestinal function, was decreased in CRC and LS174T cells. Abnormal O-GlcNAcylation contributes to the development of CRC by directly inducing the carcinogenic characteristics of cancer cells
Fernández et al[94]	2018	CRC and HEK-293T cells	ATCC	(1) Antibodies, WB, and IF. (2) Gene expression analysis. (3) Proliferation, cell viability, and invasion assays. (4) OCR and extracellular acidification rate (ECAR). And (5) IP and proteomic assay	GCNT3 can be used in stratified CRC patients with a high risk of recurrence and as a biomarker for monitoring the treatment response. The drugs that induce the expression of GCNT3 may be potential antitumor drugs for CRC. The purpose is to reduce adverse events and overcome drug resistance, which is a necessary demand for current patients and the health system
Harosh-Davidovich et al[50]	2018	CT26 murine colon carcinoma cells and NIH-3T3 murine fibroblasts	ATCC	(1) Protein extraction. β-Catenin IP. (2) Affinity purification of β-catenin with Wheat Germ Agglutinin (WGA). (3) WB. (4) Cell motility assay. (5) OGA and OGT silencing. (6) Luciferase reporter assays. (7) qRT-PCR. And (8) In vivo orthotopic mouse model of CRC	O-GlcNAcylation may enhance the proliferation and metastasis of CRC by regulating the expression of catenin and E-cadherin, which proves the influence of O-GlcNAcylation on the poor prognosis of CRC patients
Venkatakrishnan et al[95]	2017	Midsection samples of the spiral colon of the five infected pigs and healthy controls	(1) Life Technologies, Carlsbad, CA, United States. And (2) Department of Medical Chemistry and Cell Biology University of Gothenburg	(1) MUC2 and MUC5AC IF. (2) Fluorescence in situ hybridization of formalin-fixed tissue sections. (3) Mucin isolation and purification. (4) Analysis of mucin fractions. (5) Mucin sample preparation and concentration estimation. (6) Release of O-glycans from pig colon mucins. (7) PGC-LC-MS/MS characterization of O-glycans. And (8) qRT-PCR of core enzyme expression	This study provides a platform for the study of B. hyodysenteriae and its effect on the O-GlcNAcylation of mucopolysaccharide. Polysaccharides that change with infection are candidate structures that may affect the adhesion, growth, virulence gene expression, and chemotaxis
Guo et al[60]	2017	Human colon cancer cell lines LS180, HT-29, Caco-2, LS-174, SW480, and SW620	American Type Culture Collection (Manassas, VA, United States)	(1) Regulation of colon cancer stem cells and colon tumorigenesis by expression levels of O-GlcNAc. (2) Identification of O-GlcNAc-bound genes in HT-29 cells. (3) Gene expression profiling regulated by O-GlcNAc. (4) Tumor-suppressive functions of transcription factor MYBL1. O-GlcNAc epigenetically regulated. And (5) MYBL1	An epigenetic mechanism may be involved in the regulation of CCSC population and colon tumor progression through the O-GlcNAcylation level. MYBL1, a transcription activator, as a downstream target, is likely to regulate CRC progression by altering O-GlcNAcylation
Arike et al[56]	2017	(1) Mucus was scraped from the small and large intestine of ConvR and GF C57BL/6 mice. And (2) The insoluble Muc2 mucin was partially purified from duodenum, mid-jejunum, ileum, proximal colon, middle colon and distal colon by repeated 6 M guanidinium hydrochloride (GuHCl) extraction	Department of Medical Biochemistry, University of Gothenburg, Sweden	(1) Partial purification of the Muc2 mucin and its oligosaccharide analysis. And (2) Proteomics analysis of epithelial cells	There was a good correlation between the abundance of OGT and muc2-O-glycan pattern along the intestine. GF mice tend to have shorter glycans and fewer enzymes involved in glycan elongation. Compared with the mice colonized with symbiotic bacteria, the demand for glycan in GF was lower. Glycan is necessary to prevent mucin degradation, but it can also be used as a nutrient source for bacteria. However, the basic mechanism and signaling pathway of host recognizing and adapting intestinal bacteria by changing the expression of glycosyltransferase is still unclear
Lin et al[96]	2016	Surgical samples of stage III CRC patients resected	Chang Gung Memorial Hospital, Taiwan	(1) Two oxaliplatin-based regimens, mFOLFOX6 and XELOX were given postoperative treatment. And (2) GALNT14 genotyping	The GALNT14 TT genotype was associated with the T4 stage and with radical resection and adjuvant oxaliplatin chemotherapy in patients with stage III CRC. In the T4 stage, CEA > 5 ng/mL or mucus histopathology subgroup, the treatment effect was poor
Steenackers et al[19]	2016	Human CRC cell lines HT29, HCT116 and CCD841CoN	-	(1) SDS-PAGE, WB, and Antibody Staining. (2) Cell adhesion assay. (3) Proliferation assays. (4) In vitro cell Survival Assays. (5) Cell migration analysis. And (6) Confocal microscopy	The increase in O-GlcNAcylation of CRC cells gave rise to proliferation and migration of CRC cells. But the potential role and mechanism of O-GlcNAcylation in CRC transfer remain unclear
Fuell et al[97]	2015	(1) C57BL/6J WT and C57BL/6 TCRδ−/− mice. And (2) Mouse small intestine and colon tissue	C57BL/6J WT (WT; acquired from Harlan Labs) and C57BL/6 TCRδ−/− (B6.129P2-Tcrdtm1Mom/J acquired from JAX Laboratories) mice were bred and maintained as specific-pathogen free (SPF) in a conventional animal facility at the University of East Anglia	(1) RNA extraction. (2) qRT-PCR. (3) Sialic acid colorimetric assay. (4) O-glycan colorimetric assay. (5) Isolation of intestinal glycans. (6) Glycan derivatization. And (7) Analysis by MALDI-LIFT-ToF/ToF MS	The role of glycosylated proteins in the regulation of epithelial cells to limit the penetration of intestinal bacteria into the mucosa during microbial community composition changes and/or the acquisition of new organisms from the environment. It is essential to understand whether intestinal O-GlcNAcylation changes through changes in microbial communities or by signaling directly to epithelial cells

CEA: Carcinoembryonic antigen; COSMC: Core 1 β3-galactosyltransferase; CRC: Colorectal cancer; CRISPR: Clustered regularly interspaced short palindromic repeats; EMT: Epithelial-mesenchymal transition; EZH2: enhancer of zeste homolog 2; FACS: Fluorescent activated cell sorting; IF: Immunofluorescence; IHC: Immunohistochemistry; IL-13: Interleukin-13; IP: Immunoprecipitation; GALNT14: Polypeptide N-acetylgalactosaminyltransferase 14; GCNT3: Beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-N-acetylglucosaminyltransferase 3; LOH: Loss of heterozygosity; LUMC: Leiden University Medical Center; MALDI-TOF: Matrix-assisted laser desorption/ionization-time of flight; mFOLFOX6: Modified folic acid, fluorouracil, oxaliplatin 6; MS: Mass spectrometry; MYBL1: MYB proto-oncogene like 1; mTOR: Mammalian target of rapamycin; OCR: Oxygen consumption rate; OGT: O-linked N-acetylglucosamine transferase; PCR: Polymerase chain reaction; RNA: Ribonucleic acid; qRT-PCR: Real-time PCR; VUMC: VU University Medical Center; WB: Western blot; WT: wildtype; XELOX: Capecitabine combined with oxaliplatin; YY1: Yin Yang 1.

Table 2 Gene targets associated with O-GlcNAcylation in colorectal cancer

Category	Action site	Type	Pathway	Influence
Tumor suppressor gene	APC[99]	CPC; Apc cell	APC/Wnt/β-catenin signaling pathway	Affect apoptosis and growth
GT41	OGT[100]	HT29 cell	OGT silencing	Accelerate invasion and metastasis
	OGA[101]	SW480 cell	(1) OGA silencing. And (2) p53 signaling canonical Pathway	(1) Upregulate PPAR, HMG-CoA synthase, and reductase. And (2) Downregulate genes of the Akt1 substrate 1, CPT1A, AIF1, AIF2, and p53
Polypeptide N- GALNT12	T491M[102]	Germ line cell	Genetic mutations	Lead to CRC susceptibility
	T491M[102]
	R373H[102]
	R382H[102]
Programmed Cell Death Protein 1 (PDCD1)	PD-L1[81]	CD8+ cell	β-catenin/STT3 signaling pathway	Accelerate immune evasion and reduce apoptosis
GalNAc-transferases (GalNAc-Ts)	GalNAc-T3[103]	Primary cell	-	Promote differentiation and invasion
	GalNAc-T6[23]	Wild Type (WT) LS174T cell	Cell signaling pathway	Be a potential key regulator of the malignant phenotype of CRC
Tumor Necrosis Factor Receptor (TNFR)	Death Receptor (DR)-4 and DR-5[97,104]	GALNT14 genotypes and stage III Cell	(1) Apoptotic signaling pathway. And (2) DR-mediated signaling pathway	Trigger the mechanism of apoptosis.
G Protein-Coupled Receptors (GPCRs)	CXCR4 [105]	CXCR4+/− ApcMin/+ cell	(1) lncRNA XIST/ miR-133a-3p/RhoA signaling pathway. (2) JAK2-STAT3 inflammatory signaling pathway. And (3) CXCL12/CXCR4 axis	(1) Increase IL-1, IL-6, and TNF levels. (2) Invasion and Metastasis. (3) Recruit immune suppressive cells. And (4) Regulate RhoA expression by sponging miR-133a-3p
Transforming Growth Factor (TGF)	TGF-β[106]	CD44+ cell	TGF-beta signaling pathway	Enhance stem cell properties
	TWIST1[106]	CD44+ cell	TGF-beta signaling pathway	Enhance stem cell properties
	TGFβR2[107]	IBD-associated cell	p21-mediated/ TGF-β signaling pathway	Cause reduced p21 activation and reduced apoptosis
Rho	RhoA[108]	CXCR4-overexpressing HCT116 cell	RhoA/ROCK signaling pathway	(1) Regulate RhoA expression by sponging miR-133a-3p. And (2) Promote the formation of actin stress fiber and actin contractile force
Transmembrane mucins	MUC1[109]	MUC1 (+) cell	PD1/PDL1 signaling pathway.	Recruit inflammatory cytokines and evade immune surveillance.
	MUC2[110]	Muc2/Apc and ApcMin/+ cell	(1) Wnt signaling pathway. And (2) Genetic Inactivation	Contribute to the risk of developing CRC by changes in their levels
	MUC4[111]	early stage (stage I and II) cell	β-catenin signaling pathway	Predict poor survival among patients
	MUC13[112]	colitis-associated cell	β-catenin signaling pathway	Increase nuclear translocation of β-catenin and drive the development, progression, invasion and immunosuppression

CRC: Colorectal cancer; GALNT12: Acetylgalactosaminyltransferase 12; IL: Interleukin; JAK2/STAT3: Janus kinase 2/signal transducer and activator of transcription 3; MUC1: Mucin 1; PPAR: Peroxisome proliferator-activated receptor; TGF: Transforming growth factor; TNF: Tumor necrosis factor.

Table 3 Several common O-GlcNAcylation precursors

Basic structural unit	Chemical formula
Tn antigen	GalNAcα-O-Ser/Thr
Stn antigen	Siaα2-6GalNAcα-O-Ser/Thr
Core 1 (T antigen)	Galβ1-3GalNAcα-O-Ser/Thr
Core 2	GlcNAcβ1-6(Galβ1-3)GalNAcα-O-Ser/Thr
Core 3	GlcNAcβ1-3GalNAcα-O-Ser/Thr
Core 4	GlcNAcβ1-6(GlcNAcβ1-3)GalNAcα-O-Ser/Thr
LacNAc	Galβ1-3GlcNAc(Type1) or Galβ1-4GlcNAc(Type 2)
H-antigen	Fucα1-2Gal

GalNAc: N-acetylglucosamine; LacNAc: N-acetyllactosamine; STn: Sialic acid Tn.

Table 4 Therapeutic targets associated with O-GlcNAcylation and colorectal cancer

Target/Drug	Binding site/Pathway	Result
Ac-5SGlcNAc[58]	HBP	Form UDP-5SGlcNAc, and greatly decrease O-GlcNAc levels in CRC
BGJ398[59]	FGF/FGFR2 signaling pathway	Block the effect of C1GALT1 on the malignant behavior of CRC cells. C1GALT1 could be a target for treating CRC
MYBL1[60]	-	Be involved in the suppression of gene expression in CRC
sTn antigen[61]	Core 1-mediated O-glycans biosynthesis pathway	Synthesize more truncated CD44 proteins
Tn antigen[47,63]	T-synthase/COSMC pathway	Play an essential part of the design of therapeutic carbohydrate conjugate antitumor vaccine
mAbs B72.3 and CC49[113]	TAG-72	To help develop anti-Tn targeting vaccines
VVA and PNA lectins[68]	FGFR2	Targeting C1GALT1 is a promising strategy to reduce the number of CRC tumor stem cells
SIRT1[71]	GAPDH-C150	The binding of SNO-GAPDH to SIRT1 was selectively prevented in vitro
GalNAc-T6[23]	HLA class II histocompatibility antigen chain	Block the interaction between CD74 and MHC class II molecules
NHEJ inhibitors[73]	(1) HBP; (2) NHEJ	Regulate DNA repair
mAbs[74]	(1) ADCC; (2) Complement activation	Enhance the function of effector and remove certain glycan.
DC-SIGN[75]	MUC1 and CEA proteins	Improves tumor-specific T cell response and long-term tumor regression
β- catenin inhibitor KYA1797K[81]	β-catenin/STT3 signaling pathway	Reduce the stability of PD-L1, inhibit immune evasion, induce the apoptosis of Cancer stem cells, and promote the development of immunotherapy for CRC
Sunitinib[88]	NOTCH 1 signaling pathway/NOTCH 1 signaling pathway	Inhibit the expression of SW480. However, the specific regulatory mechanism needs to be further studied
Baicalin and Curcumin[88]	NOTCH 1 signaling pathway	Inhibit the proliferation and promote the apoptosis of CRC SW480 cells
CHK1/2 inhibitors	DNA damage response pathway	Undermine OGT stability and suppress replication, mitosis and cytokinesis of CRC cell
DR-5 and DR-4[97]	(1) DR-mediated signaling pathway; (2) Tenovin-6	Enhance the cytotoxic effect of oxaliplatin in CRC by up-regulating

COSMC: Core 1 β3-galactosyltransferase; CRC: Colorectal cancer; FGF: Fibroblast growth factor: FGFR2: Fibroblast growth factor receptor 2; HBP: Hexosamine biosynthetic pathway; MHC: Major histocompatibility complex; MYBL1: MYB proto-oncogene like 1; OGT: O-linked N-acetylglucosamine transferase; PNA: Peanut agglutinin; STn: Sialic acid Tn; VVA: Vicia villosa.