The self-assembly of L-histidine might be the cause of histidinemia

doi:10.21203/rs.3.rs-3091379/v1

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The self-assembly of L-histidine might be the cause of histidinemia

https://doi.org/10.21203/rs.3.rs-3091379/v1

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published 14 Oct, 2023

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L-histidine is an essential amino acid with unique biochemical and physiological properties. Histidinemia is a disease condition caused by the elevated level of L-histidine in our blood. Mutations in the histidase, an enzyme for the breakdown of histidine, is the cause of the rise in histidine concentration. To our knowledge, no research has been done on why a high concentration of histidine causes histidinemia. In this study, we provide a potential explanation why the elevated levels of histidine in the human body causes histidinemia. In this study we have found that L-histidine self-assembled in water to form nano sheet structures at physiological pH and temperature, using NMR spectroscopy and Scanning Electron Microscope techniques. The kinetics of self-assembly has been studied using real time NMR spectroscopy. We observed that both the aromatic ring and aliphatic part are equally contributing to the self-assembly of L-histidine. The symptoms of histidinemia, neurological deficits and speech delays, are similar to that of the neurodegenerative diseases caused by the self-assembly of peptides and proteins. We speculate that the self-assembly of L-histidine might be the cause of histidinemia

L-Histidine (L-His, L-H) is an essential amino acid that is used in the production of proteins and frequently present at the active site of proteins (1). It shows anti-inflammatory, anti-oxidant, and antisecretory functions within the body (2). The genetic metabolic disorder known as histidinemia is defined by the increased amounts of the amino acid histidine in our body (3). The primary signs of this disease are: (i) behavioural abnormality, which is an abnormality of mental functioning and includes a variety of affective, behavioural, cognitive, and perceptual abnormalities; (ii) hyperactivity, which is a state of being unusually or abnormally active, including in situations where it is inappropriate; (iii) moderate psychomotor retardation, which is a moderate delay in the achievement of motor or mental milestones in the development domains of a child; (iv) neurological speech impairment and (v) specialized learning disability, which are impairments of specific learning abilities like reading or writing, coordination, self-control, or attention (1).

Urocanic acid is the product of breakdown of histidine by the enzyme histidase (4). Mutation in the HAL gene, which gives instructions for producing the histidase enzyme, is the cause of the rise in histidine concentration and subsequently histidinemia (5). Increased levels of histidine are seen in the blood and urine of histidinemia patients as a result of HAL gene mutations that produce an ineffective histidase enzyme. However, no research has been done on why a high concentration of histidine causes histidinemia, despite the fact that the molecular mechanism of the increase in histidine concentration has been well researched. In this study, we provide a potential explanation how unusually elevated levels of histidine in the human body causes the histidinemia illness condition. Neurological deficits include behavioural abnormalities, hyperactivity, psychomotor retardation, speech impairment, learning disability, etc. are present in people with histidinemia (1). The symptoms of histidinemia resembles neurodegenerative amyloid disease, which develops when certain peptides and proteins self-assemble in our body and deposit as amyloid fibrils in the brain (6).

According to extensive research histidine-containing peptides and proteins can self-assemble (7). Prabhjot Singh et. al. reported the self-assembly of L-histidine in water-methanol medium (8). Using nuclear magnetic resonance (NMR) spectroscopy and scanning electron microscope (SEM) methods, we investigated the self-assembly of histidine in water, for the first time, at concentrations of 25 mM and 50 mM at physiological pH (7–8) and temperatures of 20ºC and 37ºC (physiological temperature). In this study we revealed that histidine performs self-assembly in an environment that is comparable to our physiological conditions: an aqueous medium at pH 7–8 and temperature 37°C. Real-time NMR spectroscopy was used to investigate the kinetics of self-assembly. It has been noted that the aliphatic carboxyl terminus and the aromatic imidazole ring both contribute equally to the kinetics of histidine self-assembly. The development of self-assemblies with nano-sheet morphology was confirmed by SEM images. We speculate that the self-assembly of histidine may be the source of histidinemia, a condition brought on by a larger concentration of histidine in our body that shares symptoms with neurodegenerative amyloid disorders. To the best of our knowledge, no other study of the self-assembly of L-histidine in water, its kinetics and also the reason why a higher concentration of histidine leads to histidinemia have been reported yet.

NMR spectroscopy confirmed the self-assembly of L-His in water

A very promising tool for identifying and characterising molecule self-assembly is NMR spectroscopy. The molecules in the self-assembled state have a different NMR peak position (chemical shift) than those in the free state. Figure 1 depicts the assigned 1D 1H NMR spectra of 25 mM L-His in water at pH 7–8 and 20ºC. First, we have acquired the time-dependent 1D 1H NMR spectra of 25 mM L-His in water at pH 7–8 and 20°C. Figure 2 displays an overlay of time-dependent 1D ¹H NMR spectra. We saw that the peak positions (chemical shifts) moved up field with time and stabilised at about 120 minutes (7,200 seconds). The sample solution was clear, indicating that no precipitation or aggregation has occurred. Only the self-assembly of His molecules into a well-ordered structure, in which the protons are more shielded than the free molecules, could account for the second potential cause of the chemical shift change. We obtained FE SEM images of the sample in order to understand the state of the amino acid molecules inside the solution, and we discovered that L-His self-assembled into well-ordered nanostructures (to be discussed in more detail later).

Kinetics of self-assembly of L-His in water from real time NMR

Figure 3a shows the overlay of the Ha proton peaks of the 25 mM L-His in water at pH 7–8 and temperature 20ºC and Fig. 3b displays the chemical shift values as a function of time. It is evident that a biexponential decay pathway, with a quick initial phase and a slow second phase, was followed by the change in chemical shift values. We fitted the biexponential decay equation (Eq. 1 in the materials and methods) to the plot of the variations in chemical shift as a function of time (Fig. 3b) in order to derive the rate constants for the self-assembly.

It is very interesting to observe that the Ha peak showed splitting from 10 min/600 sec onwards, indicating the existence of a different pathway of self-assembly in which the Ha proton has a different chemical environment (Figs. 3a and 3c). We have repeated the same with the peaks from other protons, Hb, Hc1, Hc2 and Hd (Figs. 4a-4h). The rate constants obtained from different protons of the L-His are tabulated in Table 1. Another interesting observation is that the rate constants from all the protons, except the second Ha peak, are the same.

It is fascinating to note that the Ha peak began to split around 10 min/600 sec, indicating the existence of a second pathway for self-assembly in which the Ha proton has a different chemical environment (Figs. 3a, 3b and 3c). With the peaks from the remaining protons, Hb, Hc1, Hc2, and Hd, we repeated the process (Figs. 4a–4h). Table 1 lists the rate constants that were found from various L-His protons. The fact that rate constants from all of the protons, with the exception of the second Ha peak, are comparable is another intriguing finding.

Effect of concentration on the rate of self-assembly of L-His in water

We conducted real-time 1D ¹H NMR experiments with 50 mM L-His in water at pH 7–8 and temperature 20ºC in order to comprehend the impact of concentration on the rate of L-His self-assembly. In Figure S1 of the Supplementary Materials, a time-dependent overlay of 1D 1H NMR spectra is displayed. We saw that the peak positions (chemical shifts) moved up field with time and stabilised at about 120 minutes. It is obvious that a monoexponential decay pathway was followed by the change in chemical shift values. We fitted the monoexponential decay equation (Eq. 2 in the Experimental Section) to the plot of the changes in chemical shift in order to derive the rate constants for self-assembly (Figure S2a-j). The rate constants that were found from various L-His protons are listed in Table 1. It is interesting to note that all of the rate constants from all the protons are comparable.

Effect of temperature on the rate of self-assembly of L-His in water

We conducted real-time NMR studies at 37°C with 25 mM and 50 mM L-His in water with a pH of 7–8 to better understand how temperature affects the rate of L-His self-assembly. Figures S3a and S3b in the supplemental materials display an overlay of time-dependent 1D ¹H NMR spectra. We saw that the peak positions (chemical shifts) moved up field with time and stabilised at about 120 minutes. It is evident that a monoexponential decay pathway was followed by the change in chemical shift values. We fitted the mono exponential decay equation (Eq. 2 in the Experimental Section) to the plot of the changes in chemical shift as a function of time (Figures S4a-j and S5a-j) in order to derive the rate constants for the self-assembly. Table 1 lists the rate constants that were found from various L-His protons. It is interesting to note that the constants from all the protons under the same circumstances are comparable.

Table 1

Rate constants of self-assembly of L- His in water at all the experimental conditions (a) 25 mM at pH 7–8 and 20°C. (b) 50 mM at pH 7–8 and 20°C. (c) 25 mM at pH 7–8 and 37°C. (d) 50 mM at pH 7–8 and 37°C.
a)	Atom	Rate constant (s ^− 1)
		k1	k2
	Ha1	0.0030	0.000026
	Ha2	0.0030	0.0003
	Hb	0.0030	0.000026
	Hc1	0.0030	0.000026
	Hc2	0.0030	0.000026
	Hd	0.0030	0.000026
b)
	Ha		0.00043
	Hb		0.00043
	Hc1		0.00043
	Hc2		0.00043
	Hd		0.00043
c)
	Ha		0.00056
	Hb		0.00054
	Hc1		0.00056
	Hc2		0.00056
	Hd		0.00055
d)
	Ha		0.00068
	Hb		0.00067
	Hc1		0.00068
	Hc2		0.00068
	Hd		0.00068

FE SEM analysis of the self-assembly of L-His in water

In the NMR spectroscopy study, the chemical shift changes as a function of time became insignificant after two hours. The scanning electron microscopy (SEM) images of 25mM L-His sample in water at a pH of 7–8 and a temperature of 20ºC were recorded for verifying the self-assembly process and for understanding the morphology of the self-assemblies. The SEM images with different resolutions are shown in Fig. 5. It is evident that histidine self-assembled into sheet-like nanostructures in water, having similar morphology as that created in a water-methanol solvent (8).

Molecules spontaneously arrange themselves into bigger structures or patterns through a process known as molecular self-assembly, without the aid of outside guidance or interference (9). The intermolecular interactions between molecules, which can be electrostatic, hydrogen bonds, van der Waals forces, or covalent bonds, are what cause this behaviour (10). Self-assembly is a fundamental process in nature that creates a variety of complex structures, including DNA helices, protein complexes, and lipid bilayers in cell membranes (11). As a potent tool for the development of novel materials with specialised features, such as nanoscale electrical devices, drug delivery systems, and sensors, molecular self-assembly is also of interest to scientists (10).

Deficiency as well as excess of amino acids in our blood cause disease conditions. Amino acids are the smallest biomolecules showing self-assembly. It has been reported that out of twenty naturally occurring amino acids ten amino acids show self-assembly which are phenyl alanine (12), tryptophan (13), histidine (13), glycine (13), alanine (13), valine (13), serine (13), isoleucine (13), proline (13) and lysine (13). Higher levels of amino acids in our blood cause a disease condition which can be generally termed as “hyperaminoacidemia” with a few exceptions like phenylketonuria (high level of phenyl alanine) and histidinemia (high level of histidine) (14, 15, 3). All these diseases as a result of high amino acid levels in our blood have the symptoms of neurological disorders like developmental delays, intellectual incapacity, seizures, mobility abnormalities etc. (13, 5, 14). It has been well studied that the mutations on the enzymes catalysing the breakdown of amino acids are responsible for the high levels of amino acids in our body (5, 14). It has been reported that high concentration of phenyl alanine in our body leads to a neurodegenerative disease called phenylketonuria, which is a consequence of self-assembly of phenyl alanine. The abnormal build-up of Tryptophan is known to cause hypertryptophanemia, a neurodegenerative disease caused by the self-assembly of tryptophan (16). In the case of all other amino acids, the molecular mechanism of diseases caused by their excess levels in our body is not well studied.

L-histidine is an amino acid that is necessary for human nutrition and has a number of medically proven advantages (2). Increased dietary histidine intake and supplementation at doses of 4.0–4.5 g histidine/d are linked to lower levels of oxidative stress, proinflammatory cytokines, fasting blood sugar, and markers of glucose homeostasis (17). Intake of histidine also enhances cognitive performance (e.g., decreases appetite, anxiety and stress, and improves sleep); potentially through the metabolism of histidine to histamine, nevertheless, the relationship between histidine and histamine in people is unclear (17). Studies have found negative effects of histidine at high intakes (> 24 g/d), including lower serum zinc levels and cognitive impairment (17).

High amounts of histidine in our blood lead to the development of a rare hereditary disorder known as histidinemia (3). People with histidinemia are unable to adequately break down histidine, thus it builds up in their blood and urine (18). The body needs histidine, an essential amino acid, to build and repair its tissues on a daily basis. Histidine and its metabolites, however, can be harmful to the body in large doses and result in symptoms like behavioural issues, speech and language problems, intellectual disability, and developmental delays (19). Histidinemia is caused by mutations in the gene that generates the enzyme histidase, which breaks down histidine, which causes histidine to accumulate (20). Histidinemia is said to be caused by an excess of histidine in the blood (3), however it has not yet been shown why a high quantity causes histidinemia. In this study, utilising NMR spectroscopy and SEM as analytical techniques, we report for the first time the self-assembly of the amino acid L-histidine in aqueous medium at physiological pH and temperature. All of these analytical methods demonstrated that L-histidine self-assembles in a condition resembling a physiological one.

The chemical shift value of a nucleus in NMR spectroscopy is dependent on its environment, hence a change in chemical shift denotes a change in environment. In the process of self-assembly, molecules are organised into certain morphologies, such as fibrils, sheets, tubes, and rods, among others. (13) Non-covalent interactions, such as hydrogen bonds, van der Waals contacts, pi-pi stacking, electrostatic interactions, and hydrophobic interactions, stabilise the self-assemblies (10). The surroundings of the monomer molecule nuclei are altered by each of these activities. Real-time 1D ¹H NMR spectroscopy has been employed in this study to better understand the self-assembly process. The self-assembly process is readily visible in our observations of the peak positions or chemical shifts of the protons in the His molecule as a function of time. It is evident that under the same experimental conditions, all of the protons followed exponential decay trajectories at rates that were comparable. By fitting exponential kinetic equations to the chemical shift changes as a function of time as stated in the methodology and results sections, it was possible to determine the rate of L-His self-assembly in water. The Ha proton peak split at a concentration of 25 mM L- His at 20 ºC, and the second peak has a different rate constant. In other words, two alternative pathways of self-assembly occur for which the Ha proton has different environments. The most crucial finding is that the self-assembly process is equally influenced by non-covalent interactions such as hydrogen bonds, electrostatic interactions, and pi-pi stacking. Both increasing concentration and temperature sped up the rates of self-assembly (see Table 1). For each proton, a biexponential route with a fast initial phase and a slow second phase was found at 25mM L-His concentration and 20ºC temperature. Since only the slow phase could be detected by NMR at higher temperatures and concentrations, comparisons are based on the rates of the slow phase that could be detected.

The scanning electron microscopy (SEM) is a very helpful tool for verifying the self-assembly process and for understanding the morphology of the self-assemblies. After two hours, the time-dependent chemical shift changes became minor, and we obtained the SEM images of the 25mM L-His sample in water with a pH of 7–8 and a temperature of 20ºC. It is obvious that histidine self-assembled into sheet-like nanostructures in water, mimicking the shape of structures created in a water-methanol solvent (8). All of the experimental methods utilised in this study, as was already discussed, demonstrated that L-histidine self-assembled in aqueous medium. This is a convincing justification for why a high level of L-histidine in human blood creates histidinemia, a condition with symptoms similar to those of other neurodegenerative illnesses.

Proteins, peptides, and amino acids self-assemble in several neurological diseases like Alzheimer's, Parkinson's, Type II diabetes, and phenylketonuria (21). Alzheimer's disease, Parkinson's disease, age-related macular degeneration (AMD), and cataracts are all diseases caused by protein self-assembly (22). The self-assembly of Aβ peptides causes Alzheimer's disease (23). Excess of amino acids in our blood leads to disease conditions having symptoms similar to that of neurodegenerative diseases. It has been well demonstrated that the diseases like phenylketonuria (excess of phenyl alanine), tyrosinemia (excess of tyrosine), hypertryptophanimea (excess of tryptophan) are arising as a result of amino acid self-assembly. For the first time we report that L-histidine can self-assemble at conditions similar to physiological condition. We speculate that the disease histidinemia, arising as a result of high level of L-histidine in our blood and having symptoms of neurological disorders, is the result of self-assembly of the L-histidine.

Data availability

All data generated or analysed during this study are included in this published article [and its supplementary information files]

Acknowledgements

We thank the Sophisticated Analytical Instrumentation Facility (SAIF) Mahatma Gandhi University, Kottayam, Kerala for the morphological studies of the self-assembly using SEM analysis.

Author contributions

Dr. Sunilkumar P. N. designed the work, interpreted the data and prepared the manuscript. Ms. Ajitha Ajikumar and Ms. Anakha Premkumar carried out the experiments, analysed the data and prepared the figures.

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Materials

The amino acid L-Histidine (with 99% purity) was purchased from NICE Chemicals Pvt. Ltd. And was directly used for preparing sample solutions in deionized Millipore water. The pH of aqueous solutions of His was measured using a pH paper (purchased from NICE chemicals Pvt. Ltd.). The deuterium oxide required for preparing NMR samples was purchased from ALDRICH. The NMR spectra were recorded using 400MHz Bruker Avance III spectrometer. High precision 5mm NMR sample tubes were used for the analysis. The NMR data were processed and analysed using Bruker Topspin 4.1.4 software (1). The time dependent chemical shift data were plotted and analysed using the Origin 6.0 software (2). Morphology images of the self-assemblies was obtained by using the Tescan MAIA3 XMH Field Emission Scanning Electron Microscope (FE SEM) instrument (3).

Methods

We have identified and characterised the self-assemblies of L-His in water under different conditions using NMR spectroscopy and FE SEM techniques. The kinetics of self-assembly was obtained from real time NMR spectra.

NMR study

The NMR analyses of L-His samples were done under four different conditions: (i) 25 mM L-His in water at pH 7-8 and temperature 20ºC, (ii) 25 mM L-His in water at pH 7-8 and temperature 37ºC, (iii) 50 mM L-His in water at pH 7-8 and temperature 20ºC and (iv) 50 mM L-His in water at pH 7-8 and temperature 37ºC. 600µL of each sample containing 5% D2O, for locking purpose, was used for recording 1D 1H NMR spectra. NMR spectroscopy is an efficient tool for understanding the self-assembly of molecules. The NMR parameter used for understanding the self-assembly is the chemical shift (δ) value, which indicates the chemical environment of an NMR active nucleus. In this study, we have recorded 100 numbers of 1D 1H spectra of the L-His solution as a function of time, the experimental time of each experiment was 5min (300 seconds) and hence the total experimental time was 8 hours 20 minutes. Initially, 1D 1H NMR spectra were recorded using the simple pulse programme ‘zg’. The solvent used was 95% H2O and 5%D2O, so the spectrum gave only a large water peak. For obtaining peaks from the sample (His) we used another pulse program, ‘zgpr’, with water suppression, which gave sample peaks with good signal to noise ratio (4). The acquisition parameters used were, spectral width (SW) 15 ppm, and number of data points (TD) 64k. The spectra were processed by applying zero filling (64k) and the window function exponential multiplication (em). All the spectra were referenced with respect to the water peak (4.8 ppm) (5).

Analysis of time dependent 1D ¹H NMR data

The chemical shifts of both aromatic and aliphatic proton peaks were picked from each spectrum using the Topspin program. The chemical shift values were plotted as a function of time using the Origin 6.0 program. The rate constants for the self-assembly of histidine was obtained by fitting the rate equation to the changes in chemical shifts as a function of time. The chemical shift changes followed biexponential and monoexponential decay pathways.

The biexponential decay function (Equation 1) used to fit on the chemical shift data is the following

δ = δ1 +A1 exp (-t/t1) + A2 exp (-t/t2)………………… (1)

Where,

δ is the chemical shift at time t

δ1 is the chemical shift at the starting time

A1 is the net change in chemical for the first phase

A2 is the net change in chemical for the second phase

t is the time

t1 is the decay time constant for the first phase.

t2 is the decay time constant for the second phase.

The rate constant for the first phase, k1 = 1/t1

The rate constant for the second phase, k2 = 1/t2

The monoexponential decay function (Equation 2) used to fit on the chemical shift data is the following

δ = δ1 +A1 exp (-t/t1) ………………… (2)

Where,

δ is the chemical shift at time t

δ1 is the chemical shift at the starting time

A1 is the net change in chemical shift during the entire process

t is the time

t1 is the decay time constant.

The rate constant k = 1/t1

FE SEM study

Significant chemical shift changes were observed during the first 2 – hour time. SEM images of the 25 mM L-His at pH 7-8 and temperature 20ºC were obtained after 2 – hour time. Sample was mounted with double sided carbon tape on aluminium stubs. The specimen was sputtered with thin layer of gold in auto fine coater and the images were examined at an accelerating voltage of 15kV. Images with 2µm, 5µm and 10µm resolutions were plotted.

References

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No competing interests reported.

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The self-assembly of L-histidine might be the cause of histidinemia

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Abstract

Figures

Introduction

Results

NMR spectroscopy confirmed the self-assembly of L-His in water

Kinetics of self-assembly of L-His in water from real time NMR

Effect of concentration on the rate of self-assembly of L-His in water

Effect of temperature on the rate of self-assembly of L-His in water

FE SEM analysis of the self-assembly of L-His in water

Discussion

Declarations

References

Methods & Protocols

Additional Declarations

Supplementary Files

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