Lectins Earlier Described As Haemagglutinins Biology Essay

Conjugated And Non Conjugated Lectin Practical

Professor George Ingram

Salford Roll No: @00263768

Manchester ID No: 78805120

Keele No: 1001853501

Module: Research Skills II – Analytical Parasitology

A. Introduction:

Lectins earlier described as haemagglutinins can be defined as sugar binding proteinaceous or glycoprotein substances that are usually of plant origin and are non-immunogenic, and has got the ability to agglutinate cells or precipitate the glycoconjugates. The agglutination occurs because the lectin has two binding sites which enable the lectin to cross-link cells via carbohydrate interaction on the cell membrane (brooks et al, 1997). They are not only of animal or plant origin, but also of viral, bacterial, fungal, and invertebrate and vertebrate form (Jacobson & Doyle, 1996). The first work done on lectins was by Hermann Stillmark in 1888 where he found agglutination of erythrocytes of human and animal, while he was working with toxicity of castor beans (Sharon & Lis, 1989). The work of Stillmark on erythrocyte agglutination from different animals was extended by Karl Landsteiner and Raubitschek (brooks et al, 1997) where he demonstrated the difference between the relative haemagglutinating activities of seeds extracts and animal red cells; as for example human erythrocytes were agglutinated by bean extracts while weakly agglutinated by lentil extracts (brooks et al, 1997, Sharon & Lis, 1989). The earlier work done was basically on plant lectins, but later the work shifted to animal lectins as well. During the 1960s, work done by Ginsberg on leucocytes from rats showed the presence of mammalian lectins.

The plant lectins are generally classified as legumes with diverse monosaccharide specificity and cereals with GlcNac/NeuAc monosaccharide specificity (Varki et al., 1999). The legume lectins are the ones which are extensively studied (Van Damme et al. 2008) and the first three dimensional structure to be studied was of Con A of legume plant lectins (De Hoff et al., 2009). Few lectins are found to be associated with endoplasmic reticulum in plant cells and they are found to be involved in surveillance mechanism for proper protein binding (banerjee et al., 2007). They are involved in protecting the plants against the invasion of micro organisms, insects and pests (De Hoff et al., 2009). The lectins are found to be increased when there is any injury or infections.

The animal lectins are classified as extracellular and intracellular lectins. The extracellular lectins comprises of calnexin family, M-type, L-type and P-type while the intracellular lectins comprise of C-type, R-type, siglecs and galectins. The extracellular lectins are involved in cellular adhesion, glycoprotein clearance and pathogen identification while the intracellular lectins are involved in various aspects of maturing glycoprotein.

Lectins are also classified according to their specificity groups, for example, mannose, galactose, N-acetyglucosamine, N-acetylgalactosamine, L-fucose and NAM.

The biological activities of lectins involve agglutination of cells or the precipitation of glycoproteins and polysaccharides. Lectins are also found to be involved in recognition of gamete and the resulting reproductive cell fusion in seaweeds (Ingram, 2004). Other functions of lectin include the mitogenic stimulation of the lymphocytes where the mitogenic lectins can stimulate a large amount of sensitized and susceptible lymphocytes irrespective of their antigenic specificity (Trowbridge, 1973). Lectins are also involved in lectin mediated killing of animal target cells like lymphocytes (cytotoxic T cells) and the sarcophaga lectins of the flesh fly have got the ability to bind with both the killer and target cells. In addition to phagocytosis of tumour cells, lectins can also cause phagocytosis of yeasts and bacteria. For example, concavalin A coated yeast cells are phagocytosed easily by mouse peritoneal macrophages.

Evangelista & Leite (Evangelista & Leite, 2002) showed the presence of specific sugar GalNAc in the midgut of Lutzomyia longipalpis by using lectin-gold conjugates. Four different lectins were used (Canavalia ensiformis [ConA], Helix pomata agglutinin [HPA], peanut agglutinin [PNA], and wheat germ agglutinin [WGA]) which were then conjugated with colloidal gold. The binding sites were found to be cytoplasmic secretory granules and microvilli of the midgut epithelium (Evangelista & Leite, 2002). The binding occurred in both sets of sand flies that were fed with fructose as well as blood meal. The reason why the peritrophic matrix of the sandfly did not bind to WGA by N-acetyl-glucosamine might be due to the complex membrane composition which does not allow the N-glycan to interact with lectin-gold conjugate.

Table 1: FITC lectin and corresponding sugar moiety

FITC-Lectin

Abbreviation

Sugar Specificity

Triticum vulgaris

WGA

NeuNAc

Canavalia ensiformis

Con A

α-CH3-mannopyranoside

Glucose/α-CH3-glucopyranoside

Arachis hypogaea

PNA

GalNAc/galactose

Lens culinaris

LCA

Mannose

Ulex europaeus

UEA-1

α-L-fucose

Bandeirea simplicifolia

BS-II

GlucNAc/glucose

Ditto

BS-I

Galactose/GalNAc

Tetragonolobus purpureas

TPA

α-L-fucose

Casaravilla et al., 2003 carried out a study in Echinococcus granulosus with the help of FITC lectins, and studied the sugar distribution on the parasite glycoconjugates. The tegument, parenchyma components and the reproductive system showed higher activity of ConA, WGA, and PNA which means that mannose is the highly expressed component of adult glycans/sugar moieties. The vaginal cells reacted only with WGA and the reproductive structures were rich in GlcNAc, N-acetyl-D-glucosamine and Gal which corresponded to the reactivity with WGA and PNA, WGA and ConA had reactivity with excretory canals. RCA reacted with tegument which corresponds to higher levels of Gal, the outcome of this study was that PNA reactivity in the reproductive system correlates to the presence of D-Gal-b-(1,3)-D-GalNAc in the terminal. The UEA I failed to bind with any FITC lectin while DBA and SBA showed low activity in the tegument.

To distinguish between the sugars present on the salivary glands of the three species of mosquitoes of Anopheles gambiae complex, FITC lectins were employed. Eight (Concanavalin A- Con A, Lathyrus odoratus- LOA, Lens culinaris, Pisum sativum-PSA, Vicia faba- VFA, Triticum vulgaris, Maclura pomifera- MPA and Ulex europaeus) out of twenty lectins reacted to salivary glands and these corresponded to higher level of mannose and N-acetylglucosamine in all the three strains of Anopheles gambiae. Further these lectins were used to distinguish between strains of Anopheles, as the level of mannose between the species was also varying. In addition, the peroxidise labelled lectins confirmed the binding specificities of the lectins detected (Mohamed & Ingram, 1993).

Schottelius (Schottelius et al, 1983) worked upon two species of trypanosome and tested them with 19 carbohydrate specific lectins. The interspecific strain differentiation of T. cruzi is possible with lectins from Euonymus europaeus, Tridacna crocea, Tridacna maxima and the human blood-group test serum anti-B from T. vespertilionis strains. T. vespertilionis agglutinated with anti-B and E. Europaeus lectins, while T.cruzi agglutinated with T. crocea and T: maxima lectins/agglutinins. This study demonstrated that N-acetylneuraminic acid is present on the surfaces of these trypanosomes.

The sugar moiety present on larval blowfly is D-mannose. This is the first sugar that has been localized in invertebrates by electron microscopy. High specificity of mannose has been shown by the lectins of peritrophic membrane of Calliphora erythrocephala. However, this lectin is only restricted to the lumen side of the midgut, while the surface of the midgut or the epithelium side of the lumen is devoid of any mannose specific lectin. Another fact that was found in this study was that the two species of bacteria (Proteus vulgaris and P.morganii) found in the lumen also have mannose specific receptors and mannose residues present on their pili. However, lectins specific for galactose are not present in the peritrophic membrane and midgut epithelium (Peters et al., 1983).

They are used directly for agglutination assays, or as substitutes for antibodies in electrophoretic techniques (Jacobson & Doyle, 1996). lectins are extensively used for glycoconjugate characterization (Zippel and Neu, 2011).

The aim of this study is to make the use of the staining properties of the lectins and use it to identify the sugar moieties present in the surface of salivary glands of the blowfly maggots. First the identification of sugar and then inhibition of carbohydrate indicates which type of sugar is present. The lectin mediated agglutination in the second half of the experiment assists in differentiating different trypanosomatid strains and thus the sugars involved in agglutination can be confirmed.

The lectin and sugar binding that takes place in the FITC conjugated lectin test is displayed by lower degree of fluorescence and the lack of specific sugars is displayed by higher degree of fluorescence. This higher degree of fluorescence is due to lack of binding.

B. Materials and Method:

Standard protocol followed – no alterations were made during the experiment.

C. Results:

The results are prepared in MS excel with the help of bar charts. The FITC conjugated lectin technique shows the amount of fluorescent lectin has bind with the tissue surface in the form of fluorescence. If the suitable sugars are present in the membrane of salivary glands, the lectin will bind to sugar and thus bind to the surface of the membrane. Thus, a comparison between fluorescent lectins binding to carbohydrate and fluorescent lectins not binding to carbohydrate can be done in the form of a graph. The fluorescence activity can be measured as:

The Fluorescence Activity

3+

intense

2+

intermediate

1+

weak to trace

0

no fluorescence

This scale measures the amount of FITC lectins binding to tissue surface as fluorescence.

fig 1: A comparison of FITC lectins binding and not binding sugar. The fluorescence activity depicts the specific sugar to the lectin present on the surface membrane of the salivary glands.

The higher levels of WGA, TPA and UEA-1 indicate higher NeuNAc and α-L-fucose. Also the level of LCA is high, which corresponds to higher level of mannose. Thus, this suggests that the level of NeuNAc and α-L-fucose is significantly higher. Also, as mentioned above, higher binding correlates to higher amount of fluorescence while less binding indicates low fluorescence.

The fluorescence activity table helps to detect the amount of fluorescence shown by the sample. A 3+ or intense fluorescence indicates presence of carbohydrates on the membrane of the salivary gland and that specific lectins bind to it. A 1+ indicates no lectin-sugar binding in the sample.

The carbohydrate present on the tissue surface can be determined by using specific carbohydrate inhibition. Peroxidise labelled lectins are used to do this. A score from o to 3+ is given where a dark brown colour indicates a positive (3+), intermediate brown colour (2+), light brown (1+), yellow-brown trace (+/-) and only yellow (0) negative.

Fig 2: figure showing the comparison of peroxidise labelled lectins to show the specific carbohydrate by using specific carbohydrate inhibition.

This figure represents the specific points of carbohydrate inhibition. The various peroxidise labelled lectins were incubated with lectin sugar solution; the presence of a sugar moiety on the surface on the salivary gland will prevent the binding of lectin to the surface. Here the lectins PNA, TPA, BS-1 and WGA binds fewer lectins, and thus have got lower colour intensity. All the lectins present in the sample have got the score of 1+ which means it is yellow brown and since this shows that the colour intensity is not high, lectin mediated binding is inhibited. Thus, this also confirms the presence of the same sugars as FITC conjugated lectins did. However, in figure 1, there was no binding between BS-1 and carbohydrate (no flourescence), while in figure 2, increased level of BS-1 have been detected, and thus counter indicating that N-acetylglucosamine/galactose might be present. With PNA, fluorescence less than 1 was detected which indicated the presence of N-acetylglucosamine/galactose on the tissue surface. These variations in the graph seen might be due to some level of biasness in the whole class data.

Detection of ConA at a higher intensity of 3+ which is very dark brown colour is the indication that the class data is not accurate.

Since, the technique used did not give accurate results; detection of surface sugar moieties with non-conjugated lectins was done where the property of agglutination was considered to detect the presence of specific sugars. In this technique, red blood cells from three species of animals, rabbit, horse and sheep was taken and agglutinated. The end point titres were identified and there seemed to be no unusual results or contamination, as all the results were according to the protocol.

Table2: represents lectins and the specific sugars inhibiting them. Titres expressed at microgram per litre.

Lectin Conc

Specific Sugar

Blood

Cell Types

expressed as mg/ml

(1 mg/ml)

moieties

Rabbit

Horse

Sheep

PAA

Glucosyl

0.000

250.000

500.000

WGA

Glucosyl

0.000

0.000

0.000

BS-II

Glucosyl

0.000

1000.000

500.000

UEA-I

Fucosyl

62.500

0.000

125.000

TPA

Fucosyl

0.000

250.000

0.000

CAA (2 mg/ml)

Neuraminyl

0.000

15.625

31.250

LPA

Neuraminyl

1000.000

0.000

250.000

VVA

Galactosyl

0.000

125.000

500.000

PNA

Galactosyl

0.000

31.250

62.500

PSA (1.25 mg/ml)

Mannosyl

0.000

78.125

625.000

LCA (1.67 mg/ml)

Mannosyl

208.750

104.375

0.000

Fig 3: figure showing agglutination of sheep horse and rabbit blood with lectins.

This figure shows the endpoint agglutination of the stock lectin. If the agglutination occurs at lower lectin concentration, there is presence of more sugar on the erythrocyte surface and vice-versa. The sugars are found to be in larger quantity in sheep blood than compared to others. Agglutination occurred at 500 g/ml titre with PSA, VVA, PAA, BS-II at well number 2 indicating mannosyl, glucosyl and galactosyl sugars are present on the sheep rbc surface. Negetive value for LCA might be due to the variation in the structures of same sugar types.

Similarly, for horse rbcs, Neuroaminyl which is the specific carbohydrate for CAA lectin and galactosyl, which is specific for PNA and VVA occurs in large quantity on the surface of the horse erythrocytes. The agglutination at lower level of lectin indicates a higher amount of sugar moieties present

For rabit rbcs, the concentration of LPA is very high in the first well and thus causes a very rapid agglutination.

Fig 4: comparison between T.congolense and T.pestani of lectin mediated agglutination. The lectins help to distinguish between the two species by detecting the specific sugar types present on the parasite cell surfaces.

This figure illustrates about how lectins can be used to distinguish between the two species of Trypanosoma by using the lectins WGA and CAA. Absence of these lectins in the graph for Trypanosoma congolense shows that glucosyl is the specific carbohydrate on T.pestani due to the presence of WGA and CAA. While, the levels of lectins TPA and LPA is high in T.congolense, which corresponds to fucosyl and neuroaminyl, is not so in case of T.pestani. Also, there may be structural variations in neuroaminyl from both the species of Trypanosoma.

Fig 5: comparison between C.lucillae and C.fasciculata of lectin mediated agglutination. The lectins help to distinguish between the two species by detecting the specific sugar types present on the parasite cell surfaces

This bar graph illustrates that on the cell surface of C.lucillae parasite, the lectin LPA binds to neuroaminyl specifically. The agglutination occurs in well number 4 which illustrates that the sugar is present in low lectin stock concentration. Also, for the lectins BS-II, LPA and LCA, the agglutination occurs at low stock lectin a concentration which corresponds to sugars glucosyl, neuroaminyl and mannosyl in C.fasciculata.

Fig 6: comparison between L.hertigi strain LV42 and L.hertigi strain LV43 of lectin mediated agglutination. The lectins help to distinguish between the two species by detecting the specific sugar types present on the parasite cell surfaces

In this chart, the identification of two different strains of Leishmania is done by identifying the specific sugar types on the surface of the parasites. Lectins such as VVA which is specific for galactosyl is present on the parasite cell type LV 42, and is present at a lower stock lectin concentration, while TPA and UEA-1 are also found on the cell surfaces of the parasites which are specific lectins for fucosyl. WGA, the specific sugar for glucosyl is present only in LV 43.

D. Discussion:

D.1: lectins that distinguish rabbit, horse and sheep RBCs:

There are many lectins that are used to distinguish the rbcs of rabbit, horse and sheep, of which few of the important ones are PAA, CAA, WA, BS-II, TPA, PSA, PNA LCA, UEA etc. WGA is found to be non-specific for all the three mammals, while LCA which is specific for mannosyl, binds only to sheep rbcs. These lectins have got specific affinity for sugar moieties present on the cell surface of these vertebrates. For example, PSA, CAA, WA, BS-II, PAA, PNA binds to their corresponding sugars in sheep and horse, where as in rabbit they doesn’t. This simple exclusion of lectins from the RBCs of rabbit automatically draw a conclusion that sugars like mannosyl, glucosyl, fucosyl are absent on the rabbit rbcs, and abundant in horse and sheep rbcs. Again, for example, the lectins UEA-1 and WA are sugar specific for sheep and rabbit RBCS but non-specific for horse red cells. The LPA is non-specific for horse rbcs, as seen from the graphs, while agglutination titre is present for the sheep and rabbit blood cells, thus confirming the presence of neuroaminyl. One TPA is found to be very specific for horse rbcs and presence of this TPA confirms the presence of fucosyl sugar moiety in horses.

D.2: lectins that distinguish T.pestani and T.congolense:

The lectins used to distinguish between these two species of Trypanosomes are WGA, CAA, TPA and LPA. LPA which corresponds to neuroaminyl is specific for T.pestani and absent in T.congolense. Also, the levels of TPA is higher in T.congolense than T.pestani corresponds to the carbohydrate fucosyl.

WGA and CAA, which are specific lectin for glucosyl, are abundant in T.pestani, and they act specifically for this parasite because agglutination occurs quite late for these lectins, which indicates tht the sugar is present in abundant qualities.

D.3: lectins that distinguish C.luciliae and C.fasciculata:

The lectins used to distinguish between C.luciliae and C.fasciculata is LPA, LCA, BS-II. The sugar moeities that are specific for C.luciliae are glucosyl and neuroaminyl which corresponds to the lectins BS-II and LPA at very low concentrations. The sugar neuroaminyl is present in very large quantity as compared to glucosyl, which can be easily inferred from the graph. The carbohydrates present on C.fasciculata are mannosyl, glucosyl, neuroaminyl, galactosyl fucosyl, of which the later two are found in very less concentration, which is confirmed by the lectin agglutination time. The agglutination that occurs with LPA specifically indicates C.luciliae, as the agglutination time is faster with a high end point.

D.4: lectins that distinguish between Leishmania hertigi strains Lh42 and Lh43:

The sugar moieties present on the surface of the parasites are galactosyl and fucosyl. The lectins used to distinguish between these two strains of parasites are VVA, TPA and UEA-I. The agglutination titre endpoints for these three lectins are 15.625mg/ml, 3.906mg/ml and 15.65 mg/ml respectively for UEA-I, TPA and VVA. These end point agglutination titres are variable for LV42 and LV43, and thus being helpful to distinguish between these two strains. For LV43, end point agglutination titre never reaches the lower stock concentrations and WGA is a specific lectin for LV43 which corresponds to the sugar glucosyl.

E. The best lectin that could be used to distinguish between Trypanosome, Leishmania and Crithidia:

The lectin to distinguish leishmania from Trypanosoma and Crithidia would be either VVA,TPA, UEA-I and PNA and the agglutination is noticed to occur in very low stock concentrations and the corresponding sugars are present on the surface of the parasite.

For trypanosome species, the best lectin is CAA which causes agglutination of the surface sugars.

For Crithidia species, the best lectins are BS-II and LCA and they correspond to the sugar mannosyl, which is abundant in the parasite and they are found to agglutinate at very low stock concentrations.

F. A comparative assessment of fluorescent, enzyme and agglutination techniques:

Fluorescent technique is considered quite a successful technique when the assessment is being done by an expertise person. As because, the rating of the level of fluorescence depends upon the experience of the individual. This technique has been used for many years to identify the presence of sugar moieties on the surface of the cells. This technique is found to be excellent for direct labelling methods especially for the cell suspensions and the results obtained are generally dramatic and photogenic. Also, the test is very sensitive and double labelling is also possible (Brooks et al., 1997). Also, it is used for detailed mapping of the molecules in the cells and tissues by the method of multiple fluorescent staining (Suzuki et al., 2007). Larger size of the peroxidise molecule can dramatically alter the carbohydrate binding site while, FITC being a smaller molecule less likely alters the carbohydrate binding site. FITC labelled lectins are good for quantitative population studies and cell function assays, as the fluorescent lectins have got the ability to sort the cells that can express the sugars. An approach to reduce the error of rating of level of fluorescence is the use of computer programme packages. The results obtained in this way are more reliable and valid. FITC are very efficient in detecting and grading malignant tumours as this employs the method of cell surface interactions and also histochemical examinations.

Mentioning the disadvantages, the reliability of the technique on the individual’s ability to make a decision sometimes can be considered as its disadvantage. Also, the fluorescence technique can be ephemeral; which means if the samples are left out after staining with fluorescent dyes; it may fade off quite soon (Brooks et al., 1997). To overcome this, photographing of the slide is helpful. Also, this technique is highly sensitive to pH changes and this may give rise to broad spectrum nature of fluorescence. However, this method is relatively easy to perform and does not require any intensive process. It is a bit time consuming process because of the fact that the fluorescent lectins need time to incubate for the sugars and the lectins to interact.

If the fluorescence is more, it may shine out like stars and may affect the visibility of the sample and thus making it a bit difficult to identify the exact cell type. This may lead to interpretation of false results. One method to overcome this problem is the use of phase contrast microscopy (Brooks et al., 1997).

The fluorescent and enzyme labelling techniques are not as accurate as the lectin mediated agglutination technique. This might be due to the reason that agglutination is the main character of the lectins. However, problems may arise when two or three strains of parasite may bind to the same lectins. Other laboratory factors may also alter the results (Sharon & Lis, 1989). Lectin agglutination methods are useful for detecting the pathological processes like tumours and malignant transformations.

Lectin (Abbreviation)

Peroxidase Unconjugated - with sugar

FITC Conjugates - with sugar

WGA

2

2

Con A

2.9

0

UEA-I

3

2

BS-I

0.9

0.5

TPA

0.9

1

PNA

0.9

0.5

Table2: Comparision of compares FITC conjugates and peroxidase non-conjugates (conjugated with sugar)

Fig7: graph comparing the FITC conjugate with peroxidise unconjugates. With sugar

Lectin (Abbreviation)

Peroxidase Unconjugated - without sugar

FITC Conjugates - without sugar

WGA

3

3

Con A

4

0.9

UEA-I

1

0.5

BS-I

1.9

0.5

TPA

1

0.9

PNA

2

2

Table3: compares FITC conjugates and peroxidase non-conjugates (without sugar)

Fig 8: graph comparing the FITC conjugate with peroxidise unconjugates. Without sugar

The tables and graphs are the comparison of the fluorescent conjugates with peroxidise unconjugates with and without sugar. Salivary glands are found to be binding more to Con A and this was confirmed by both FITC and peroxidise, thus can be interpreted that glucose and N-acetylneuraminic acid are present in the salivary glands of blow fly.

G: Conclusion:

The test performed for detection of sugars with the help of lectins certainly detected sugars that were present on the surface of the salivary gland of the blow fly and the blood samples from different animals. Although there was an error in the results (UEA-I), rest of the data came out almost the way it should have been. The detection of carbohydrates or sugar moieties with their linking to lectins and then the inhibition with the help of enzymes certainly helped in detection of the desired sugars and thus helped in knowing the sugars present in various cell types.