Purification and characterization of a novel haemagglutinin from Chlorella pyrenoidosa

DOI 10.1007/s10295-006-0145-9

O R I G I N A L P A P E R

Puri

Wcation and characterization of a novel haemagglutinin

from Chlorella pyrenoidosa

Received: 7 June 2005 / Accepted: 3 May 2006 / Published online: 15 June 2006 © Society for Industrial Microbiology 2006

Abstract We previously identiWed a strong haemag-glutination activity in the freshwater unicellular green alga, Chlorella pyrenoidosa. Here, we sought to purify and characterize the haemagglutinin associated with this activity. Ammonium sulfate precipitation, gel Wltration on sephacryl S-200 and DEAE-Sepharose ion-exchange chromatography were used to purify the haemagglutinin, which was designated CPH (Chlorella pyrenoidosa haemagglutinin). The molecular weight of CPH was estimated as 58 kDa by SDS-PAGE and 60 kDa by gel Wltration of the native protein, indicating that this haemagglutinin exists as a monomer. The hae-magglutinin activity of CPH was inhibited by glycopro-teins, especially yeast mannan, but not by monosaccharides or disaccharides, indicating that CPH is carbohydrate-speciWc. In addition to the composition of CPH shown to be rich in glycine and acidic amino acids, heamagglutinating activity of CPH was insensi-tive to variations in pH or the presence of divalent cat-ions, and atomic force microscopy revealed that the protein is rod-shaped. These results indicate that the characteristics of CPH are consistent with its identi Wca-tion as a haemagglutinin, and suggest that CPH may be a viable candidate for applications in a variety of bio-medical Welds.

Keywords Haemagglutinin · Chlorella pyrenoidosa · PuriWed · Rod-shaped

Introduction

The haemagglutinins, which are widely distributed in nature, are carbohydrate-binding proteins associated with important eVects, such as cell aggregation and gly-coconjugate precipitation. As such, haemagglutinins can be used as carbohydrate probes, making them use-ful tools in a variety of biochemical and biomedical research areas. Although the physiological function of agglutinins in algae is not clear, biochemical studies indicate that algal haemagglutinins have potentially useful applications in biochemical, drug, and clinical studies. For example, several studies have reported that algal haemagglutinins exhibit immunomodulatory and antitumour activities in vitro and in vivo [16, 19, 22].

Since Boyd et al. [3] Wrst reported identiWcation of marine algae haemagglutinin, many researchers have investigated the haemagglutinating capacities of marine algae extracts [1, 7, 8, 10, 24], and several algal haemagglutinins have been isolated to date [2, 16, 26]. The majority of previous studies have focused on hae-magglutinins from marine macroalgae. Unfortunately, the practical use of marine macroalgal haemagglutinins are limited by some critical problems, including few scientists study this subject, low concentrations of mol-ecules in algal extracts, and some high haemagglutinat-ing activity algae are diYcult to collect [16, 25].

Recent works have suggested that these limitations may be overcome by the use of microalgae, which are more easily cultured and manipulated [4, 18, 27, 30]. To date, haemagglutinins have been isolated from the C. -Y. Chu · L. -P. Ling (&)

Institute of Microbiology and Biochemistry, National Taiwan University,

Taipei 106, Taiwan

e-mail: Sping.bbs@yahoo.com.tw

C. -Y. Chu · R. Huang

Institute of Oceanography, National Taiwan University, Taipei 106, Taiwan

freshwater microalgae Microcystis aeruginosa (M228), M. viride [29, 30], and Oscillatoria agardihii [27]. How-ever, only a few studies have examined the possible applications of haemagglutinins from freshwater mic-roalgae.

Although only few attempts have so far been made at freshwater microalgal haemagglutinin, we still believe that microalgae haemagglutinins could com-plement faults of macroalgal haemagglutinins and have wide application in many Welds. In a recent screening for haemagglutinating activity of freshwater microalgae, we found that Chlorella pyrenoidosa dis-played a strong and stable activity [4]. The aim of this study was to purify and characterize this novel hae-magglutinin from C. pyrenoidosa as one of a series of studies of agglutinins and their activities focusing on the microalgae.

Materials and methods Cells and culture conditions

The C. pyrenoidosa cells used in this study were iso-lated from the river in Taiwan, and were grown and maintained on proteose medium [14] composed of KNO₃ (0.25 g L¡1), MgSO₄•7H₂O (75 mg L¡1), K₂PO₄ (0.175 g L¡1), NaCl (25 mg L¡1), CaCl₂ (10 mg L¡1), Fe solution (1 ml L¡1_{), A5 solution (1 ml L}¡1_{), and} prote-ose peptone (1 g L¡1). The pH was adjusted to 6.0 before the medium was autoclaved at 121°C for 20 min. Algae were maintained at 25°C in 1.2 L containing 1.0 L culture under 80
mol m¡2S¡1 illumination with a 12 h light:12 h dark cycle. Cells were cultured for 10– 14 days, and then harvested at the stationary phase by centrifugation at 10,000£g for 20 min. Harvested cells were lyophilized and stored at ¡20°C until use.

Preparation of extracts from harvested cells

Lyophilized Chlorella cells (10 g) were resuspended in 200 ml of Tris–HCl buVered saline (TBS; pH 7.4, con-taining 25 mM NaCl) at 4°C, and sonicated (Virsonic, USA) in an ice bath for ten 3 min cycles Each mixture was then centrifuged at 15,000£g for 20 min, and the supernatant was used as the extract for haemagglutinin puriWcation.

Haemagglutination assay

Human B erythrocytes were used to purify haemag-glutinin as previously reported [9]. The erythrocytes were concentrated and washed three times with

phos-phate-buVered saline (PBS; pH 7.2, containing 0.1 M NaCl), collected by centrifugation, and diluted 1:50 PBS. This erythrocyte suspension was used directly in the agglutination activity assay, using serial two-fold dilutions as previously reported [7]. Algal extracts were placed in wells (50
L to which equal volumes of human B erythrocyte suspension were added. The plates were gently shaken, and left for 2 h at room temperature. The reciprocal of the highest dilution exhibiting positive haemagglutination was expressed as the extract titer.

PuriWcation of haemagglutinin from C. pyrenoidosa The C. pyrenoidosa cellular extracts were precipitated with increasing percentages of ammonium sulfate. Precipitates showing positive haemagglutination reac-tions (primarily from the 40–60% ammonium sulfate fractions) were pooled, dissolved in distilled ion-exchanged water, and then dialyzed against TBS (con-taining 50 mM NaCl) at 4°C for 24 h. During dialysis, the buVer was refreshed once every 4 h. The dialyzed sam-ples were centrifuged, and the supernatants were sub-jected to gel Wltration chromatography on a sephacryl S-200 column (26£60 cm; Pharmarcia) previously equili-brated with TBS and eluted at a Xow rate of 1.0 ml min¡1. The eluted protein fractions were detected their haemagglutining activity by human B erythrocyte. Samples showing positive reactions were collected and dialyzed against TBS (containing 50 mM NaCl) at 4°C for 24 h. Each dialyzed fraction was centrifuged and the supernatant was subjected to ion-exchange chromatogra-phy using a DEAE-Sepharose column equilibrated with the same buVer and eluted by stepwise increases in NaCl concentration up to 0.5 M at Xow rate of 1.0 ml min¡1_. The active fractions were collected and dialyzed against TBS (containing 50 mM NaCl). Finally active elutes were dialyzed against distilled ion-exchanged water, lyophi-lized, stored in buVer at ¡20 C, and considered to be puriWed haemagglutinin (designated as CPH). SDS-PAGE and gel Wltration methods were used for molecu-lar weight determination using thyoglobulin (Mw 670 kDa), bovine gamma globulin (Mw 158 kDa), oval-bumin (Mw 44 kDa), and Myoglobin (Mw 17 kDa) as markers (Pharmacia; Uppsala, Sweden).

Protein and sugar contents

Protein contents were quantitated by bovine serum albumin as the standard according to the Lowry’s [20] method. Sugar contents were quantitated by phenol-sulfuric methods [6] with D-galatose as the standard.

Amino acid composition

Amino acid composition of the puriWed protein was analyzed by an Amino Acid Analyzer (Schimazu, SCL¡10 A). The protein sample was hydrolyzed with 6N HCl in an evacuated tube at 110°C for 24 h. The hydrolyzed sample was then applied to the analyzer for amino acid composition.

Haemagglutination-inhibition test

PuriWed CPH with an activity titer of eight was tested for vulnerability to inhibition by various carbohydrates and glycoproteins according to the methods of Hori et al. [9]. The utilized carbohydrates and glycoproteins were obtained from Sigma Co (USA), and included the mono-saccharides L(¡)arabinose, L(-)fucose, D(+)glucose, D(+)galactose, D(+)mannose, D(+)xylose and N-acetyl-D-galactosamine, the disaccharides lactose, D(+)maltose and D(+)sucrose, and the glycoproteins -globulin, asia-lofetuin, fetuin, mucin, and yeast mannan.

Testing the eVect of pH and divalent cations on CPH activity

The eVects were determined following the methods used by Hori et al. [9], using B erythrocytes for activity assay. For examination of the eVect of pH on haemag-glutination activity, each 1.0 mL of puriWed CPH was dialyzed against 0.1 M buVer solutions with pH values ranging from 3.0 to 11.0 at 4°C for 24 h, followed by thoroughly dialysis against PBS to rule out the eVect of pH on the activity assay. The buVers used were: citrate buVer (pH 3.0–4.0), acetate buVer (pH 4.0–5.0), phos-phate buVer (pH 6.0–8.0), Tris-HCl buVer (pH 8.0–9.0) and carbonate buVer (pH 10.0–11.0). For divalent cat-ions, 1.0 ml of protein solution was mixed with or with-out 10 mM EDTA plus an equal volume of 5 mM CaCl₂, ZnCl₂, MgCl₂ or MnCl₂. Each mixture incu-bated at room temperature for 2 h, centrifuged, and then assayed.

Morphology of CPH

For observing the image of CPH, the puriWed protein was attached through electrostatic interactions by plac-ing it in contact with freshly cleaved mica that had been coated with poly L-lysine, a positively charged com-pound. After cleaning the mica with methanol and Milli-Q water, 5
L of 10¡2M poly L-lysine solution was applied and incubated for 30 min. The mica sur-face was then washed with Milli-Q water before intro-duction of the puriWed CPH specimens. Specimens of

5
L were applied onto the poly L-lysine-treated mica for 5 min at room temperature, followed by washing with distilled water and drying in air prior to the Atomic force microscope (AFM) experiments.

Atomic force microscope (AFM) experiments in tapping mode of operation were carried out using scan-ning probe microscope (NT-MDT, Russia). Commer-cial silicon cantilevers nano-probe with a spring constant 0.03 and 0.1 N m¡1 were used for tapping modes in this study. Tapping mode images were col-lected in a broad range of frequencies, 10–20 kHz. The tip of the silicon nitrite probe being 10 nm in diameter, the default gain value 10 nA, and data acquisition and image processing software an integral part of each scanning probe microscope from NT-MDT.

Electrophoresis

The puriWed protein was fractionated by SDS-poly-acrylamide gel electrophoresis (SDS-PAGE) [13] on 12.5% gels. Samples and standards were prepared in 10% SDS with 1% 2-mercaptoethanol at 100°C for 5 min prior to application to electrophoresis. The sepa-rated proteins were stained with Coomassie brilliant blue R¡250 for 60 min. Proteins (Mark 12™_{, Novex) of} known molecular weights (14; 20; 30; 43; 67; 94 kDa) were used as reference proteins.

Results

Isolation of a haemagglutinin from C. pyrenoidosa Crude protein extracts from C. pyrenoidosa were pre-cipitated with increasing quantities of ammonium sul-fate and puriWed with a two-step chromatographic puriWcation procedure. During ammonium sulfate pre-cipitation, the highest haemagglutinating activity was present in fractions precipitated with 40–60% ammo-nium sulfate (maximum haemagglutination titer = 212), with the 0–20% and 60–80% fractions showing moder-ate activity and the 20–40, and 80–100% fractions showing negligible activity (Fig.1). Notably, most of the algal proteins precipitated in the 40–60% ammo-nium sulfate range.

The 40–60% ammonium sulfate fraction was sub-jected to gel Wltration on a Sephacryl S-200 column, yielding peaks in both activity and protein absorbance at fractions 46–55. The active fractions were collected and further puriWed by ion-exchange chromatography on a DEAE-Sepharose column eluted by increasing concentrations of NaCl. Haemagglutination activity was detected in the fractions eluted with 0.1 M NaCl,

and the eluted haemagglutinin was designated CPH (C. pyrenoidosa haemagglutinin). The results of puriW-cation are summarized in Table1, where approxi-mately 926 mg protein was extracted from 10 g of C. pyrenoidosa cells (dry weight). The total protein and speciWc activity of CPH was 1.45 mg and 1544.83 (titer mg¡1_{) from the Wnal DEAE ion-exchange step,} respectively. The puriWed haemagglutinin was approxi-mately 27.7-fold more active than that from the extracts.

Chemical properties of CPH

SDS-PAGE and gel Wltration were used to determine the molecular weight of puriWed CPH. A single band at 58 kDa was detected on SDS-PAGE (Fig.2), while a single symmetrical peak at 60 kDa was obtained by molecular exclusion chromatography of the native pro-tein on a Sephacryl S-200 column (Fig.3), indicating that CPH exists as a monomer. Furthermore, the

neu-tral sugar content of CPH with the phenol-sulfate method was not determined.

The amino acid composition of CPH (Table2) pre-dominantly consists of glycine (18.3%), aspartic acid/ asparagine (14.8%), and glutamic acid/glutamine (16.4%). The content of acidic amino acids was fairly larger than that of basic amino acids, i.e., histidine, lysine and arginine. The puriWed protein also con-tained a relatively small quantity of sulfur-containing amino acids, such as methionine and cystine shown in Table2.

Carbohydrate binding speciWcity

The binding speciWcity of CPH for carbohydrates was detected using a haemagglutination inhibition test, which revealed that the haemagglutination activity of CPH was not inhibited by any of the tested monosac-charides or disacmonosac-charides (Table3), whereas all Wve tested glycoproteins showed inhibitory eVects. Yeast Fig. 1 Ammonium

sulfate-precipitation steps of haemag-glutinin from C. pyrenoidosa. Protein concentration (bars) and haemagglutinating activ-ity (circles) were detected from various steps. Haemag-glutination activity was ex-pressed as a titer, the reciprocal of the highest two-fold dilution exhibiting posi-tive haemagglutination

Ammonium sulfate conc (%)

-20 0 20 40 60 80 100 120 Protein conc (mg/mL) 0 2 4 6 8 10 12 14 Hemagglutin ating activ ity (log 2 titer) 0 3 6 9 12 15 18 21 24

Table 1 PuriWcation of CPH from C. pyrenoidosa

Haemagglutinating active (HA) was expressed as titer, and the titer is reported as the inverse of the last dilution with positive aggluti-nation against human B-type erythrocytes

a _{Total haemagglutination titer (HA £ vol)}

PuriWcation step THAa _Total

protein (mg)

SpeciWc activity

(titer mg¡1₎ PuriWcation _(fold)

Yield (%)

Crude extract 51,600 926 55.72 1.00 100

40–60% of (NH4)2SO4 ppt 61,440 190 323.37 5.80 119

Gel Wltration chromatography 3,840 10.6 362.26 6.50 7.44

mannan showed the strongest inhibitory eVect, fol-lowed by mucin, asialofetuin and fetuin, with globulin showing the weakest inhibitory eVect.

EVects of pH and divalent cations on CPH activity The agglutinating activity of CPH was fairly stable over pH 4–11, but was slightly reduced at a pH of 3. CPH activity was not signiWcantly aVected by the presence of divalent cations such as Ca2+, Mg2+, Mn2+ and Zn2+, with or without EDTA. These results indicate that the CPH-directed haemagglutination reaction is indepen-dent of divalent cations. In addition, the Fig.4 shows the two dimension atomic force micrographs of CPH from C. pyrenoidosa. The result of atomic force micro-graphs shows the morphology of CPH as rod-shaped.

Discussion

The Chlorella species are widespread in the air and soil, as well as in fresh and salt water. These simple uni-cellular algae reproduce asexually via non-motile autospores, grow quickly and have highly eVective photosynthesis. These characteristics make Chlorella a good choice for use in the lab [31]. In addition, Chlo-rella cells are rich in protein and contain special func-tional compounds, making them an ideal health food source [17, 23, 28]. Thus, scientists have sought to investigate Chlorella cellular biology for both labora-tory and commercial applications.

We previously examined the haemagglutination activity of extracts from 44 species of freshwater micro-algae and identiWed C. pyrenoidosa as having a high haemagglutination activity [4]. In previous works, marine algal haemagglutinins/lectins have been iso-lated by salt precipitation followed by gel Wltration and ion-exchange chromatography [11, 16]. Here, we sought to purify and characterize the relevant protein using chromatography of ammonium sulfate-tated cell extracts. We found that the fraction precipi-tated with 40–60% ammonium sulfate had increased agglutination activity than the raw extract, suggesting that salt precipitation is useful for removal of inhibi-tory compounds from the algal extracts. However, we also found that the haemagglutination activity decreased following the gel Wltration step, suggesting that this portion of the puriWcation process could be improved for better yield of active CPH.

Previous reports have revealed that the freshwater microalgal haemagglutinins/lectins are monomeric pro-teins with high aYnities for glycopropro-teins but not monosaccharides [27, 29, 30]. These molecules contain high percentages of acidic amino acids, and show no dependence on metal ions for their haemagglutination activities. SpeciWcally, Oscillatoria agadihii agglutinin (OAA) was shown to be a monomer protein with a Fig. 2 Determination of the molecular weight of CPH from

C. pyrenoidosa by gel Wltration. The Mw standards (Bio-Rad)

were used Thyroglobulin (670 kDa); Bovine gamma globulin (158 kDa); Chicken Ovalbumin (44 kDa), and Myoglobin (17 kDa). The molecular weights on the vertical axes are logarith-mic function of the original value, and the predicted CPH molec-ular weights are indicated on the plot

0 0.5 1 1.5 2 2.5 3 3.5 35 40 45 50 55 60 65

Fraction number (No.)

L

og Mw

Fig. 3 SDS-PAGE of hemagglutinin from C. pyrenoidosa and

molecular weight standards. Lane 1 shows molecular weight stan-dards, which are composed of phosphorylase b (94 kDa), Bovine serum albumin (67 kDa), Ovalbumin (43 kDa), Carbonic anhy-drase (30 kDa), and Trypsin inhibitor (20 kDa)

58 94 67 43 30 20 kDa 1 2 kDa

strong aYnity for yeast mannan and no cation depen-dency for its haemagglutination activity [27], while a lectin from the cyanobacterium, Microcystis aeruginosa (M228), was found to be a monomer protein with a molecular weight of 57–72 kDa and a particular aYnity for galactose and N-acetyl-galactosamine [29]. In this work, the molecular weight of CPH was estimated to be 58 kDa by SDS-PAGE and 60 kDa by gel-Wltration, suggesting that CPH exists as a monomeric protein with no disulWde bonds. Furthermore, CPH had aYni-ties for glycoproteins but not monosaccharides or disaccharides, and its haemagglutination activity was

cation independent. Collectively, these characteristics indicate that CPH should be classiWed as a freshwater microalgal lectin.

The interaction of proteins with biomaterial surfaces plays a key role in the host response to implanted devices [15, 21]. Algal haemagglutinin/lectin molecules are considered potentially useful in biochemical and clinical applications, due to their carbohydrate-binding speciWcities [5, 12, 25]. However, the precise physiolog-ical role(s) of these proteins are not yet known. A number of recent reports have suggested that algal haemagglutinin/lectin receptors present on the surfaces of marine microalgae may function in cell recognition, cell surface adhesion, symbiosis with marine inverte-brates or other algae, and phagocytosis of virus and bacteria [12]. To provide additional insight into our novel haemagglutinin molecule, we recently examined CPH using atomic force microscopy (AFM), which has been used to observe the morphologies of biological macromolecules such as proteins, antibodies, and DNA fragments [15].

AFM images revealed that CPH is a rod-shaped protein. In the future, it will be interesting to use AFM to examine conformational changes in CPH during gly-coprotein adsorption or desorption; these studies may provide new insights into microalgal physiology, pro-tein structure, and the function of the CPH/carbohy-drate interaction. Finally, we recently developed a CPH-speciWc monoclonal antibody. Immunogold label-ing and electron microscopy revealed that CPH is localized beneath the surface layer of the cell, between the cytoplasm and cell membrane (unpublished data). Thus, we hypothesize that CPH is widely distributed on the Chlorella cell surface and may function in cell recognition, adherence, symbiosis, and/or may play additional yet-unknown physiological roles.

Table 2 Amino acid composition of CPH

a _{ND means not detected} b _{Asx means Asp and Asn} c

Glx means Glu and Gln

Amino acid Mol. % Amino acid Mol. %

Asxb _{14.8 Val 4.5} Thr 4.9 Met 1.0 Ser 3.1 Ile 3.2 Glxc _{16.4 Leu 5.0} Pro 4.8 Tyr 3.8 His 6.1 Phe 2.0 Ala 5.8 Gly 18.3

Cys (half) ND a _{Lys 3.1}

Arg 2.7

Table 3 Haemagglutination-inhibition with carbohydrates and

glycoproteins

a

The minimum inhibitory concentration that is required to inhibit completely the haemagglutinating activity of a titer, 8

b _{“–” indicates no inhibitory activity at the concentration of} 100 mM carbohydrates Carbohydrate and glycoproteina Minimum inhibitory concentration (
g mL¡1₎ Monosaccharide L-arabinose –b L-fucose – D-glucose – D-galactose – D-mannose – D-xylose – N-acetyl-D-glucosamine – Disaccharide – Maltose – Sucrose – Lactose – Glycoprotein -Globulin (bovine) 125 Asialofetuin 62.5 Fetuin 62.5 Mucin 31.3 Yeast mannan 15.6

Fig. 4 AFM image of C. pyrenoidosa heamagglutinin (CPH) on

In sum, we herein puriWed and characterized a novel haemagglutinin from C. pyrenoidosa with pH stability, good carbohydrate speciWcity and high-level activity, making it a strong candidate for future applications in lectin research and biomedical applications.

Acknowledgments We wish to acknowledge the Wnancial

sup-port for this study received from the National Science Council (Taiwan) (NSC: 92–2317-B-002–030).

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