Introduction

Carbohydrates make up a vital and ubiquitous class of essential nutrients and play important roles in innumerable biological processes, including cell recognition, intercellular signal transduction, cell growth, and cell differentiation (1,2,3). However, progress toward understanding their structure and function lags behind other biomolecules, such as nucleic acids and proteins. This is primarily due to the high degree of complexity and heterogeneity displayed by carbohydrate-containing materials. Today, methods available for characterizing complex carbohydrates include spectroscopy [mass spectroscopy (MS), Fourier transform infrared, Raman, and nuclear magnetic resonance (NMR) spectros-copies], immunochemical labeling, electron microscopy, and X-ray crystallography. These approaches often have low spatial resolution (micron level or above) or require extensive sample preparation that may alter the molecular structure of the polysaccharide sample.

The plant cell wall is an intricate conglomerate that includes numerous polysaccharides as major components (e.g., cellulose, xyloglucan, xylans, mannans, arabinans, and pectins). The synthesis and incorporation of the various wall polysaccharides into a durable fibrous network has been the subject of numerous recent investigations (4). For this purpose, various fluorescence and electron microscopic labeling methods have proved especially effective. In this context, immunocytochemical (5,6,7,8), enzyme-(9,10,11,12,13,14), and lectin-based (9,15) labeling techniques have been developed to examine the in situ distribution of plant cell wall polysaccharides.

Carbohydrate-binding modules (CBMs) are noncatalytic protein modules found in many carbohydrate-hydrolyzing enzymes, such as the cellulases and hemicellulases. They function as recognition modules that convey the catalytic modules of these enzymes to the target substrate. Several hundred CBMs have been identified to date, and these have been grouped into 45 families using amino acid sequence similarity algorithms (afmb.cnrs-mrs. fr/CAZY/index.html). The structures and ligand specificities of many CBMs have also been determined experimentally (16). Three types of CBMs have now been proposed based on their structure and function; these include type-A, which bind to specific surfaces of insoluble, crystalline cellulose and/or chitin; type-B, which bind to individual glycan chains; and type-C, or small sugar-binding CBMs, which have lectin-like properties and bind to mono-, di-, or trisaccharides.

The fine-tuned polysaccharide recognition capacity of the CBMs has prompted their development as precise molecular probes for mapping the chemistry and structure of carbohydrate-containing materials (16,17,18,19). CBMs have thus been used for fluorescence labeling of plant cell walls (20,21,22) and for ultrastructural labeling of Valonia cellulose crystals (23). Notably, following the original submission of this paper, differential recognition of plant cell walls in different tissues by different xylan-specific CBMs was reported (22). However, these published descriptions of CBM probes require complex labeling procedures or the development of monoclonal antibodies. In addition, imaging at the nanometer scale with high resolution has not been described.

In the present study, we describe an approach that simplifies the labeling procedures, requires minimal sample preparation, and heightens substantially the resolution power. Three CBMs from three different families that possess three distinctive polysaccharide specificities were engineered with dual His-tags to facilitate their coordination to highly luminescent quantum dots for correlative imaging using fluorescence and electron microscopy. In addition, the CBMs were each expressed as fusion proteins, with either green fluorescent protein (GFP) or red-fluorescent protein (RFP), to allow direct imaging using fluorescence microscopy at the near-molecular level of resolution. This approach complements newly developed techniques for visualizing the interaction of CBMs with carbohydrate composites and is potentially capable of single molecule detection.

Materials and Methods

Sample Preparation

Crystalline cellulose was prepared from the algae, Valonia ventricosa, as described previously (24,25). V. ventricosa vesicles were harvested and rinsed with distilled water, then were boiled in 2% NaOH for 6 h (changing fresh 2% NaOH every 2 h). The purified vesicles were neutralized with dilute HCl and washed with distilled water. For the preparation of cellulose crystals, purified vesicles were homogenized for 10 min at 10,000 rpm. The resulting suspensions were hydrolyzed by stirring for 5 h in 3.5 M HCl and then neutralized with a few drops of NaOH solution and washed with distilled water. The suspensions of Valonia cellulose crystal were stored at 4°C in distilled water containing 0.01% (w/v) NaN₃. Small aliquots of cellulose crystal stock suspension were diluted in 20 mM Tris buffer, pH 8.0, to make the final concentration of 1 mg/mL used for this study.