Spatial N-glycomics of the human aortic valve in development and pediatric endstage congenital aortic valve stenosis

https://doi.org/10.1016/j.yjmcc.2021.01.001Get rights and content

Highlights

  • Pediatric endstage congenital aortic valve stenosis involves unknown regulation of glycosylation.

  • N-glycans, metabolic sugar codes, are spatially defined in the young valve structure.

  • Altered N-glycan processing occurs within the disorganized, diseased valve structure.

  • N-glycans contribute to human aortic valve structure and are disease regulated.

Abstract

Congenital aortic valve stenosis (AS) progresses as an obstructive narrowing of the aortic orifice due to deregulated extracellular matrix (ECM) production by aortic valve (AV) leaflets and leads to heart failure with no effective therapies. Changes in glycoprotein and proteoglycan distribution are a hallmark of AS, yet valvular carbohydrate content remains virtually uncharacterized at the molecular level. While almost all glycoproteins clinically linked to stenotic valvular modeling contain multiple sites for N-glycosylation, there are very few reports aimed at understanding how N-glycosylation contributes to the valve structure in disease. Here, we tested for spatial localization of N-glycan structures within pediatric congenital aortic valve stenosis. The study was done on valvular tissues 0–17 years of age with de-identified clinical data reporting pre-operative valve function spanning normal development, aortic valve insufficiency (AVI), and pediatric endstage AS. High mass accuracy imaging mass spectrometry (IMS) was used to localize N-glycan profiles in the AV structure. RNA-Seq was used to identify regulation of N-glycan related enzymes. The N-glycome was found to be spatially localized in the normal aortic valve, aligning with fibrosa, spongiosa or ventricularis. In AVI diagnosed tissue, N-glycans localized to hypertrophic commissures with increases in pauci-mannose structures. In all valve types, sialic acid (N-acetylneuraminic acid) N-glycans were the most abundant N-glycan group. Three sialylated N-glycans showed common elevation in AS independent of age. On-tissue chemical methods optimized for valvular tissue determined that aortic valve tissue sialylation shows both α2,6 and α2,3 linkages. Specialized enzymatic strategies demonstrated that core fucosylation is the primary fucose configuration and localizes to the normal fibrosa with disparate patterning in AS. This study identifies that the human aortic valve structure is spatially defined by N-glycomic signaling and may generate new research directions for the treatment of human aortic valve disease.

Introduction

Congenital aortic valve stenosis (AS) occurs at an incident rate of over 13.9 in 1000 births, yet there are no medicinal treatments for the disease [1,2]. AS progresses as an obstructive narrowing of the aorta due to congenital defects that fuse, enlarge and deform the aortic valve (AV) leaflets [3,4]. Nearly all patients with moderate to severe AS develop left ventricular hypertrophy leading to heart failure [5]. AS has two primary endpoints that are age-dependent, implicating different disease mechanisms [6,7]. In pediatric endstage AS, excess and disorganized extracellular matrix is deposited within the leaflets leading to pediatric heart failure. In adult endstage AS, leaflets are also enlarged by ECM expansion, but intervention to prevent heart failure is not required until middle age when calcific nodules emerge to restrict heart function [6,8]. Surgical valve replacement is the only treatment for AS, and for pediatric endstage AS, this is especially detrimental. Pediatric valve replacement devices are limited by their inability to grow with age [9] and increased mortality rates [10]. A major knowledge gap in developing therapies inhibiting pediatric AS is that the molecular mechanisms remain poorly defined. In particular, multiple glycoproteins associated with progression and endstage AS may have N-linked glycans (N-glycan) [[11], [12], [13], [14], [15]], yet these important post-translational modifications remain mostly undefined in human heart valve biology.

N-glycosylation is a complex carbohydrate post-translational modification that modulates protein signaling during development and disease. The N-glycan structure is composed of a series of sugar units created from nucleotidic, metabolic, and translational processing [[16], [17], [18], [19]]. N-glycans attach to the glycoprotein structure at asparagine residues within the consensus sequence NXS/T>>>C, ≠P [[20], [21], [22]]. All eukaryotic N-glycans have a common core of two N-acetylglucosamine (GlcNAc) and three mannose residues [16]. Further sugar units are added through enzymatically driven activities of trimming, branching and capping dependent on biological status [16,23]. Generally, genetic mutations affecting assembly or initiation of the N-glycan structure are embryonic lethal, whereas mutations associated with trimming, extension, and capping produce variable abnormalities [17]. Disease-mediated alteration of N-glycan structures results in inappropriate cell-cell recognition, migration and proliferation, and disease-enhancing changes along major signaling pathways such as TGFβ1 [[24], [25], [26], [27]], JNK [28], ERK [29], EGFR [30], Notch [31].

There are many N-linked glycoproteins that regulate the aortic valve structure and function during development and disease. The glycoproteins tenascin C (TNC), tenascin X precursor (TNXB), Von Willebrand Factor (VWF), and fibronectin 1 (FN1) are regulated with AS progression and all have multiple sites for N-glycosylation [[11], [12], [13], [14], [15]]. Collagens are the main proteins of the valve scaffold [13,[32], [33], [34], [35]] and all contain consensus sites for N-glycosylation. Numerous studies have reported spatial and cell specific N-glycosylation changes associated with cardiac pathologies [[36], [37], [38], [39], [40], [41], [42], [43]]. However, there are few studies on N-glycan regulation of the aortic valve structure. During normal aortic valve aging, regulation of N-glycosylation was independent of the glycoprotein carriers [44]. This study demonstrated that the normal adult AV from 21 to 51 years of age has sequential increases in high mannose structures, sialic acids, tri-antennary branching with decreases in core fucosylation. Adult endstage AS has been linked to decreased lumican N-glycosylation with multiple sites of N-glycosylation varying in ECM type proteins [45]. Although it is apparent that dynamic changes in N-glycosylation are important to AV heath and disease, details on N-glycan structure in development or pediatric disease remain unknown.

Spatial regulation of the aortic valve structure is critical for valvular function [35]. During early somatic growth, the AV develops a precise trilayer extracellular matrix structure that becomes deregulated in disease, and this leads to heart failure [4,35,46]. However, understanding spatial regulation of N-glycan patterns within the valve structure is an analytical challenge. Conventional studies on N-glycan regulation use liquid chromatography coupled to mass spectrometry [47,48]. These studies require homogenization of the tissue which hinders spatial investigations. Imaging mass spectrometry (IMS) is an approach that allows investigation of the spatial localization of molecules within the tissue microenvironment [[49], [50], [51]]. IMS scans across tissue, collecting thousands of x, y datapoints or pixels, each of which report up to thousands of analytes. Each analyte may be visualized as a heatmap of expression across the tissue referenced to tissue features.

Here, we present the first report of N-glycan configuration and regulation involved in pediatric congenital aortic valve stenosis. Pediatric normal and AS valvular tissue sections were probed for N-glycan profiles using high mass accuracy matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). N-glycan configuration was evaluated using new technological advances that define N-glycan structural configuration while maintaining spatial references to tissue features. Specifically, an on-tissue chemical modification was optimized to report localization of sialic acid linkages within valve structures [52]. New enzymatic approaches were optimized to define aortic valve N-glycans with core versus branched fucose structures [52]. Parallel RNA-Seq analysis was used to evaluate glycosidase and glycosyltransferase regulation associated with N-glycosylation changes. Our results suggest that N-glycosylation is spatially regulated within the normal valve structure with a dominant core-fucose phenotype, and that pediatric endstage AS involves increases in certain sialylated N-glycans independent of age. This study defines role for N-linked glycosylation in human aortic valve development and childhood disease which may lead to new research directions for the treatment of human heart valve disease.

Section snippets

AV tissue

Tissues were obtained from the Vanderbilt Cardiology Core Laboratory for Translational and Clinical Research (CLTCR) and from the National Disease Research Interchange. The aortic valve tissues were collected under the Vanderbilt Pediatric Congenital Heart Disease Biorepository, written consent was obtained and the project was approved by theVanderbilt Institutional Review Board (IRB) and the IRB at the Medical University of South Carolina. Aortic valves were obtained from excision of leaflet

Overview

A collection of 19 aortic valve tissues was tested for localization and regulation of N-glycosylation using novel advances in N-glycomic tissue imaging. We describe the novel technology for glycomics spatial referencing with the results. Data from each tissue includes Movat's pentachrome staining, imaging mass spectrometry data of N-glycans and additional derivatization and endoglycosidase studies accompanied by antibody and lectin staining. Results are shown as 2D images ordered by

Discussion

N-glycosylation is developmentally regulated and disease altered within the human aortic valve structure. Our study found that the trilayer structure of the normal valve is defined by very specific N-glycan structures. In AS, N-glycans are not only altered by abundance and configuration but also show distinct spatial patterns throughout the valvular structure. Spatial patterns may attributable to cellular phenotypes or changes in metabolism as part of the underlying congential disease.

Conclusion

One of the greatest challenges in understanding heart valve biology is that structure regulates function and function feeds back to regulate structure. Molecular regulation across the valvular subanatomy is thus critical to integrity of the valve structure and heart function. New Omics methods for spatial referencing have been developed within the last decade that may be used to test and report molecular regulation in combination with hemodynamics in single cells, humans and animal models [49,67

Disclosures

None.

Acknowledgements

Funding provided specifically for this work by the American Heart Association (16GRNT31380005) to PMA with additional support by the NIH/NIGMS (P20 GM103542) and National Center for Advancing Translational SciencesUL1 TR000445, which supported initial studies for the project. PMA is enormously grateful to HSB for early career mentoring on valve development and biology. CLC supported by HL007260 (NIH/NHLBI). Support to RRD provided NCI/IMAT (1R21CA207779) and by the South Carolina Centers of

References (119)

  • N. Shen et al.

    Inhibition of TGF-β1-receptor posttranslational core fucosylation attenuates rat renal interstitial fibrosis

    Kidney Int.

    (2013)
  • L. Li et al.

    Inhibiting core fucosylation attenuates glucose-induced peritoneal fibrosis in rats

    Kidney Int.

    (2018)
  • X. Wang et al.

    Core fucosylation regulates epidermal growth factor receptor-mediated intracellular signaling

    J. Biol. Chem.

    (2006)
  • H. Allam et al.

    The glycosyltransferase GnT-III activates Notch signaling and drives stem cell expansion to promote the growth and invasion of ovarian cancer

    J. Biol. Chem.

    (2017)
  • L.E. Dupuis et al.

    Altered versican cleavage in ADAMTS5 deficient mice; A novel etiology of myxomatous valve disease

    Dev. Biol.

    (2011)
  • R. Zhang et al.

    Reducing immunoreactivity of porcine bioprosthetic heart valves by genetically-deleting three major glycan antigens, GGTA1/β4GalNT2/CMAH

    Acta Biomater.

    (2018)
  • C. Ashwood et al.

    Reference glycan structure libraries of primary human cardiomyocytes and pluripotent stem cell-derived cardiomyocytes reveal cell-type and culture stage-specific glycan phenotypes

    J. Mol. Cell. Cardiol.

    (2020)
  • X. Wen et al.

    Inhibiting post-translational core fucosylation prevents vascular calcification in the model of uremia

    Int. J. Biochem. Cell Biol.

    (2016)
  • M. Przybyło et al.

    Age effect on human aortic valvular glycoproteins

    Arch. Med. Res.

    (2007)
  • A.I. Saeed et al.

    TM4 microarray software suite

    Methods Enzymol.

    (2006)
  • M.L.A. De Leoz et al.

    High-mannose glycans are elevated during breast cancer progression

    Mol. Cell. Proteomics

    (2011)
  • H.J. An et al.

    Glycomics and disease markers

    Curr. Opin. Chem. Biol.

    (2009)
  • A. Varki

    Sialic acids in human health and disease

    Trends Mol. Med.

    (2008)
  • Z. Yu et al.

    Identification and characterization of S2V, a novel putative siglec that contains two V set Ig-like domains and recruits protein-tyrosine phosphatases SHPs

    J. Biol. Chem.

    (2001)
  • N. Mitra et al.

    SIGLEC12, a human-specific segregating (pseudo) gene, encodes a signaling molecule expressed in prostate carcinomas

    J. Biol. Chem.

    (2011)
  • P.M. Lackie et al.

    Expression of polysialylated N-CAM during rat heart development

    Differentiation

    (1991)
  • K.J. Rodriguez et al.

    Regulation of valvular interstitial cell phenotype and function by hyaluronic acid in 2-D and 3-D culture environments

    Matrix Biol.

    (2011)
  • A.C. Liu et al.

    The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology

    Am. J. Pathol.

    (2007)
  • K. Ohtsubo et al.

    Glycosylation in cellular mechanisms of health and disease

    Cell

    (2006)
  • M.C. Lucena et al.

    Epithelial mesenchymal transition induces aberrant glycosylation through hexosamine biosynthetic pathway activation

    J. Biol. Chem.

    (2016)
  • J. Li et al.

    Unmasking fucosylation: from cell adhesion to immune system regulation and diseases

    Cell Chem. Biol.

    (2018)
  • R.B. Hinton et al.

    Extracellular matrix remodeling and organization in developing and diseased aortic valves

    Circ. Res.

    (2006)
  • C.M. Otto et al.

    Aortic-valve stenosis—from patients at risk to severe valve obstruction

    N. Engl. J. Med.

    (2014)
  • B.W. McCrindle et al.

    Are Outcomes of Surgical versus Transcatheter Balloon Valvotomy equivalent in neonatal critical aortic stenosis?

    Circulation

    (2001)
  • A. Vincentelli et al.

    Acquired von Willebrand syndrome in aortic stenosis

    N. Engl. J. Med.

    (2003)
  • A. Della Corte et al.

    Spatiotemporal patterns of smooth muscle cell changes in ascending aortic dilatation with bicuspid and tricuspid aortic valve stenosis: focus on cell–matrix signaling

    J. Thorac. Cardiovasc. Surg.

    (2008)
  • K.-I. Matsumoto et al.

    Noticeable decreased expression of tenascin-X in calcific aortic valves

    Connect. Tissue Res.

    (2012)
  • P. Stanley et al.

    N-Glycans

  • A. Varki et al.

    Glycans in development and systemic physiology

  • E. Bieberich

    Synthesis, processing, and function of N-glycans in N-glycoproteins

  • R.D. Marshall

    The nature and metabolism of the carbohydrate-peptide linkages of glycoproteins

    Biochem. Soc. Symp.

    (1974)
  • M.S. Lowenthal et al.

    Identification of novel N-glycosylation sites at noncanonical protein consensus motifs

    J. Proteome Res.

    (2016)
  • E. Bause

    Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes

    Biochem. J.

    (1983)
  • K.W. Moremen et al.

    Vertebrate protein glycosylation: diversity, synthesis and function

    Nat. Rev. Mol. Cell Biol.

    (2012)
  • H. Lin et al.

    Blocking core fucosylation of TGF-β1 receptors downregulates their functions and attenuates the epithelial-mesenchymal transition of renal tubular cells

    Am. J. Phys. Renal Physiol.

    (2011)
  • C.-F. Tu et al.

    FUT8 promotes breast cancer cell invasiveness by remodeling TGF-β receptor core fucosylation

    Breast Cancer Res.

    (2017)
  • G. de Vreede et al.

    A Drosophila tumor suppressor gene prevents tonic TNF signaling through receptor N-glycosylation

    Dev. Cell

    (2018)
  • K. Kovács et al.

    2-deoxy-glucose downregulates endothelial AKT and ERK via interference with N-Linked glycosylation, induction of endoplasmic reticulum stress, and GSK3β activation

    Mol. Cancer Ther.

    (2016)
  • S. Shapiro et al.

    Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of D-aspartate and nuclear weapons-related radiocarbon

    J. Clin. Investig.

    (1991)
  • S. Balasubramanian et al.

    mTOR in growth and protection of hypertrophying myocardium

    Cardiovasc. Hematol. Agents Med. Chem.

    (2009)
  • Cited by (15)

    View all citing articles on Scopus
    View full text