Understanding the Biological Rationale for the Diversity of Cellulose-directed Carbohydrate-binding Modules in Prokaryotic Enzymes

Abstract

Plant cell walls are degraded by glycoside hydrolases that often contain noncatalytic carbohydrate-binding modules (CBMs), which potentiate degradation. There are currently 11 sequence-based cellulose-directed CBM families; however, the biological significance of the structural diversity displayed by these protein modules is uncertain. Here we interrogate the capacity of eight cellulose-binding CBMs to bind to cell walls. These modules target crystalline cellulose (type A) and are located in families 1, 2a, 3a, and 10 (CBM1, CBM2a, CBM3a, and CBM10, respectively); internal regions of amorphous cellulose (type B; CBM4-1, CBM17, CBM28); and the ends of cellulose chains (type C; CBM9-2). Type A CBMs bound particularly effectively to secondary cell walls, although they also recognized primary cell walls. Type A CBM2a and CBM10, derived from the same enzyme, displayed differential binding to cell walls depending upon cell type, tissue, and taxon of origin. Type B CBMs and the type C CBM displayed much weaker binding to cell walls than type A CBMs. CBM17 bound more extensively to cell walls than CBM4-1, even though these type B modules display similar binding to amorphous cellulose in vitro. The thickened primary cell walls of celery collenchyma showed significant binding by some type B modules, indicating that in these walls the cellulose chains do not form highly ordered crystalline structures. Pectate lyase treatment of sections resulted in an increased binding of cellulose-directed CBMs, demonstrating that decloaking cellulose microfibrils of pectic polymers can increase CBM access. The differential recognition of cell walls of diverse origin provides a biological rationale for the diversity of cellulose-directed CBMs that occur in cell wall hydrolases and conversely reveals the variety of cellulose microstructures in primary and secondary cell walls.Cellulose, a major component of plant cell walls and of the biomass of the earth, is a chemically invariant polymer comprising of up to 10,000 ␤-1,4-linked-glucosyl residues. In cell walls cellulose is found in the form of linear insoluble microfibrils that result from self-association of cellulose chains subsequent to synthesis by plasma membrane-located complexes. Biophysical studies have shown that cellulose microfibrils can exist in highly ordered crystalline, semi-ordered para-crystalline, and disordered noncrystalline (amorphous) states (1). Cellulose microfibrils extensively encircle plant cells and are intimately associated with other polysaccharide networks in dense composites that comprise primary and secondary cell walls. The orientation of cellulose microfibrils and their connections with other wall polysaccharide networks are key factors in the generation of cell and organ shapes and mechanical strength during plant growth and development (2).The interactions between cellulose chains both within microfibrils and with matrix polysaccharides within the plant cell wall restrict their accessibility to enzyme attack. To overcome this problem glycoside hydrolas...

SALT-OVERLY SENSITIVE5 Mediates Arabidopsis Seed Coat Mucilage Adherence and Organization through Pectins

“…We used two carbohydrate-binding modules (CBMs; CBM3a and CBM28) in parallel with S4B staining to further examine the distribution of cellulose in the adherent mucilage. CBM3a binds preferentially to crystalline cellulose structures, whereas CBM28 binds preferentially to amorphous cellulose structures (Blake et al, 2006;Dagel et al, 2011). In wild-type adherent mucilage, there are obvious differences in the staining pattern of S4B and CBM3a ( Fig.…”

“…JIM13, MAC207, JIM8, and CCRC-M7 (CarboSource) have also been previously described (Bradley et al, 1988;Pennell et al, 1989Pennell et al, , 1991Knox et al, 1991;Puhlmann et al, 1994;Yates and Knox, 1994;Steffan et al, 1995;Yates et al, 1996;Majewska-Sawka and Nothnagel, 2000;Pattathil et al, 2010). CBM3a, specific to more crystalline cellulose, and CBM28, specific to more amorphous cellulose regions (Blake et al, 2006), were treated as primary antibodies in identical solutions before treatment with mouse anti-histidine (Qiagen). Goat anti-rat secondary antibody conjugated to AlexaFluor488 was used against JIM5, JIM7, JIM8, JIM13, and MAC207, whereas goat anti-mouse conjugated to AlexaFluor488 (Molecular Probes; Invitrogen) was used as a tertiary antibody against the CBMs, CCRC-M36, and CCRC-M7.…”

CELLULOSE SYNTHASE-LIKE A2, a Glucomannan Synthase, Is Involved in Maintaining Adherent Mucilage Structure in Arabidopsis Seed

“…The Deposition of Crystalline Cellulose Is Altered in csla2-1 Mucilage Heteromannan and crystalline cellulose was shown previously to be distributed in specific patterns within adherent mucilage (Blake et al, 2006;Sullivan et al, 2011; Lee et al, 2012). To further examine whether the spatial distribution of cellulose in adherent mucilage was modified in csla2-1, whole mount immunolabeling with probes that detect heteromanan and cellulose was carried out on mature dry seeds.…”

“…There are also minor amounts of pectic HG in the adherent mucilage, with high methylesterified HG in the external domain compared with the internal domain of the adherent layer (Willats et al, 2001;Macquet et al, 2007a;Rautengarten et al, 2008;Sullivan et al, 2011;Saez-Aguayo et al, 2013). In addition, the adherent mucilage contains cellulose (Blake et al, 2006;Macquet et al, 2007a), which is entangled with RG-I and is thought to anchor the pectinrich mucilage onto seeds (Macquet et al, 2007a; HarpazSaad et al, 2011 HarpazSaad et al, , 2012Mendu et al, 2011;Sullivan et al, 2011). As such, Arabidopsis seed mucilage is considered to be a useful model for investigating the biosynthesis of cell wall polysaccharides and how this process is regulated in vivo (Haughn and Western, 2012).…”

Analysis of the physical properties of developing cotton fibres

“…CBMs, with distinct molecular recognition capacities, have been used to locate in situ the presence of crystalline and amorphous regions of cellulose in plant materials [57]. However, such biological techniques have never been used to quantify molecular changes in cotton fibres during development, yet they offer a new method of qualitative and quantitative measurement of changes to the structure of cellulose that occur during the development of cotton fibres.…”