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Originally published In Press as doi:10.1074/jbc.M405664200 on August 16, 2004

J. Biol. Chem., Vol. 279, Issue 44, 45441-45449, October 29, 2004
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Structure Elucidation of a Novel Yellow Chromophore from Human Lens Protein*

Rongzhu Cheng{ddagger}, Qi Feng, Ognyan K. Argirov, and Beryl J. Ortwerth

From the Mason Eye Institute, The University of Missouri, Columbia, Missouri 65201

Received for publication, May 20, 2004 , and in revised form, August 16, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and 1H, 13C, and two-dimensional NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and fluorescence at 410 nm when excited at 343 nm. Analysis of the purified compound by reversed-phase HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure to be 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium, a cross-link between the {epsilon}-amino groups of two lysine residues, and a five-carbon ring. Because this cross-link contains two lysine residues and a dihydropyridinium ring, we assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests were made using a high-performance liquid chromatograph equipped with a diode array detector. These measurements revealed a significant enhancement of K2P in cataract lens proteins (613 ± 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 ± 51 pmol/mg of WS protein) when compared with aged normal human lens proteins (261 ± 93 pmol/mg of WISS protein or 23 ± 15 pmol/mg of water-soluble (WS) protein). These data provide chemical evidence for increased protein cross-linking during aging and cataract development in vivo. This new cross-link may serve as a quantitatively more significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The aging of human lens is characterized by increasing levels of water-insoluble (WI)1 proteins in association with high levels of yellow chromophores and non-tryptophan fluorescence (1–4). These biochemical changes are all enhanced in senile and brunescent cataractous lenses. Glycation of lens proteins has been suggested to be a major protein modification in older lenses, and indeed diabetic patients have an increased risk of cataract formation (5). The human lens is largely composed of elongated fiber cells that are derived from epithelial cells located in a thin layer on the anterior surface. The innermost layers of the lens are formed during embryonic development and remain throughout life (6). Due to the lifelong stability of the lens crystallins, they can continuously accumulate damaging modifications. In time, these modifications may be responsible for the protein aggregation and protein cross-linking seen in age-onset cataracts (7–9). Furthermore, a dramatic, visible increase in protein-bound chromophores occurs during brunescent cataract formation, especially in tropical countries (10, 11). However, the chemical mechanisms responsible for the development of lens coloration and cataract remain unknown.

In the past two decades, enormous progress has been made in our understanding of the Maillard reaction and its contribution to age-related changes and complications of diabetes. Maillard chemistry is initiated by the non-enzymatic reaction of reducing sugars (12), dicarbonyl compounds or the degradation products of ascorbic acid (13, 14) with proteins. The intermediates, either Schiff bases and/or Amadori compounds, then undergo a series of further reactions through dicarbonyl intermediates to form advanced glycation end-products (AGEs) (15). A major consequence of AGE accumulation is the formation of covalently cross-linked proteins, especially in long-lived tissue proteins. AGE cross-links may thus contribute to the pathophysiological changes seen in aging, cataract formation, and the long term complications of diabetes (16, 17). Although some cross-links, such as pentosidine (18, 19), LM-1 (vesperlysine A) (20, 21), MOLD/GOLD (22, 23), and glucosepane (24), have been detected in human lens tissues, their quantities are rather low even in cataracts. Therefore, the major cross-links may not have been isolated, possibly because they do not survive acid hydrolysis of the proteins.

In this study, we report the isolation and characterization of a novel lysine-lysine cross-link, 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium (K2P), from enzymatic digests of aged and cataract human lens proteins. In addition, quantitative determination of this cross-link in individual lenses was performed in both normal human lenses and brunescent cataract lenses. The age-dependent increase of this cross-link in normal human lenses and its significant enhancement in cataract lenses support the possibility that glycation plays an important role in lens proteins insolubilization and precipitation during aging and cataract development.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All reagents were obtained in the highest purity available from Sigma. De-ionized water (18-megohm resistance or greater) was used for all experiments. Phosphate buffers were treated with 10 g/liter Chelex resin (200–400 mesh, Bio-Rad) overnight to remove trace metal ion contaminants as described by Beyer and Fridovich (25), and filtered through a 0.2-µm nitrocellulose filter before use.

Normal human lenses were obtained as a kind gift from the Heartland Lions Eye Tissue Bank (Columbia, MO) following cornea removal. All the lenses were clear, without obvious cataracts, and the eyes were screened for infectious diseases. The lenses were stored individually, frozen at –70 °C until use. Brunescent cataracts were collected from hospitals in Rajkot, India, and stored frozen at the Patney Eye Clinic in Rajkot, India. They were transported by hand to Columbia, MO, where they were stored individually, frozen at –70 °C until use.

Preparation of Lens Proteins—Aged normal human lenses and early stage brunescent cataract lenses (dark yellow to tan in color) were decapsulated, pooled, and homogenized in deionized water (1 ml per lens) with a hand Dounce homogenizer. The resulting homogenate was centrifuged at 27,000 x g for 20 min. The 27,000 x g pellet was obtained and washed three times with an equal volume of deionized water. These supernatants were combined and designated the WS fraction. The final pellet was resuspended again in deionized water and sonicated in ice for 5 min in a Heat Systems-Ultrasonics Inc. Sonicator (Model W-375) at a power setting of 4 and a duty cycle of 40%. The solubilized protein was recovered after centrifugation at 27,000 x g, and the pellet was resuspended and sonicated again. The second solubilized supernatant, when combined with the first, represented 95–100% of the total WI protein and was designated the WI sonicate supernatant (WISS), as described previously (26–28).

Newborn calf lens proteins were used as a control and prepared from the outer cortex of thawed, decapsulated lenses by removing the outer 1.0 cm of tissue with a cork borer. This tissue was homogenized in deionized water and centrifuged at 27,000 x g. The supernatant was dialyzed extensively against 5.0 mM Chelex-treated phosphate buffer (pH 7) and used directly. The protein content was determined using the BCA assay as described by the manufacturer (Pierce, Rockford, IL).

Proteolytic Digestion of Aged Normal Human Lens or Cataract Lens Proteins—Proteolytic digestion was carried out by a modification of the method of Luthra et al. (29). Typical preparations contained protein from 150 to 200 aged or cataract human lenses, each protein fraction was adjusted to 10 mg/ml in 5.0 mM Chelex-treated phosphate buffer, pH 7.0, and 3.6 mg of porcine intestinal peptidase (Sigma P7500) was added per 100 mg of protein. The digestion mixture was then sterile filtered into a sterile tissue culture flask, bubbled with argon, and incubated for 24 h at 37 °C in the dark. After 24 h, a solution of Pronase (Sigma P5147) was added through a syringe filter, to a level of 3.6 mg of Pronase per 100 mg of protein. Digestion was continued for 72 h, during which Pronase was added two additional times after 24 and 48 h, respectively. Following adjustment of the digest to pH 8.5 with 1.0 M NaOH, leucine aminopeptidase (Sigma L 5658) was added to a final concentration of 40 units per 100 mg of protein. The digest was sterile filtered, bubbled with argon, and incubated for 24 h at 37 °C. After adjusting the pH of the digest to 7.6 with 1.0 M HCl, it was sterile filtered again, bubbled with argon, and further proteolysis was carried out by the sequential sterile addition of 5.0 mg of trypsin per 100 mg of protein (Sigma T8642), 5.0 mg of chymotrypsin per 100 mg of protein (Sigma C4149) followed 24 h later by the addition of 3.6 mg of proteinase K (Sigma P6556) per 100 mg of protein. Calf lens protein control and corresponding enzyme blanks incubated without added protein were also incubated and analyzed on several occasions. Amino acid analysis was used to evaluate the efficiency of the enzyme digestion by comparing the total free amino acids in a 12% sulfosalicylic acid supernatant to that of an equivalent aliquot acid hydrolyzed in 6.0 N HCl, for 20 h.

Isolation and Purification of K2P from Human Lenses—WI lens proteins were separated from 150 pooled aged normal human lenses and from 350 types I and II cataract lenses from India, these were classified based on the extent of browning of the lens, and subjected to extensive enzymatic digestion. The digest was applied to Bio-Gel P-2 size exclusion chromatography, and the early eluting peak at 330 nm (Peak 1) was concentrated and multiple aliquots subjected to HPLC. The final product was judged pure by virtue of a single ninhydrin-positive spot that corresponded to a single UV and fluorescent spot on Silica Gel 60 F254 aluminum medium thin layer chromatography (Merck) and a single UV HPLC peak under various chromatographic conditions.

Bio-Gel P-2 Size-exclusion Chromatography—The total digest from ~2 g of either aged or cataract WISS protein was concentrated and subjected to gel filtration chromatography on a Bio-Gel P-2 column (5 x 76 cm, bed volume 1.5 liter), with 25 mM formic acid as eluant. Fractions of 14.5 ml were collected at a flow rate of 1 ml per minute and monitored for absorbance at 330 nm, for fluorescence at excitation/emission = 350/450 nm, and for amino acid content by the fluorescamine assay (30). An enzymatic digest of calf lens protein (control) was run under the same conditions. Peak fractions were pooled according to the A330 readings. Five peaks (designated P1–P5) were obtained, and peak 1 was used for the isolation of the novel cross-link in this study.

High-performance Liquid Chromatography—Peak 1, isolated from several pooled aged and cataract WISS protein digests by Bio-Gel P-2 column, was concentrated and injected into a preparative C18 reversed-phase column (Prodigy 21.2 x 250 mm, Phenomenex, Torrance, CA) using a Shimadzu HPLC system (Shimadzu Scientific Instruments, Inc., Lenexa, KS) with Model LC-6AD pumps, an SCL-10A controller and an SPD-M10A photodiode array detector. The column was eluted for 2 min with 5% (v/v) acetonitrile in water containing 0.1% (v/v) heptafluorobutyric acid (HFBA), at a flow rate of 8 ml per minute, and then with a linear gradient from 5% to 45% (v/v) acetonitrile in 0.1% HFBA over 40 min, followed by a linear increase to 100% acetonitrile over the next 5 min. The eluant from the column was monitored with an on-line diode array detector.

One peak, which had a maximum absorbance at 343 nm, was collected and further purified over a semi-preparative reversed-phase Prodigy column (10 x 250 mm). The column was eluted for 2 min with 25 mM formic acid-water (buffer A), at a flow rate of 1.8 ml per minute, and then with a linear gradient to 25% buffer B (water/acetonitrile = 1/1, v/v, with 25 mM formic acid) over 25 min, followed by a linear increase to 50% buffer B over the next 8 min and continued to 100% buffer B over the next 5 min. The eluant from the column was monitored with an on-line photodiode array detector. The A343 peak was concentrated and purified once again with the same solvent system but with an analytical Prodigy RP-HPLC column. The final purification was achieved by using an analytical-reversed phase Prodigy column (4.6 x 250 mm) with the same solvents and gradient as the preparative HPLC mentioned above. After extensive drying with a speed vacuum, 1.3 mg of K2P was obtained.

Spectroscopy—Absorption spectra were recorded with a Cary 1E spectrophotometer (Varian Analytical Instruments, Walnut Creek, CA) connected to a Dell PC computer. Fluorescence spectra were recorded with a Hitachi F-2500 Fluorescence spectrophotometer (Hitachi Instruments Inc.).

Samples for NMR spectroscopy were dissolved in 300 µl of H2O/D2O (9/1, v/v) or 100% D2O after two exchanges with D2O (Cambridge Isotope Laboratories Inc.) and transferred to a 5-mm Advanced Microtube (Shigemi, Allison Park, PA) and scanned for 1H NMR, 13C NMR, DEPT, COSY, HMQC, and HMBC at 25 °C with a Bruker (Karlsruhe, Germany) model ARX-500 MHz spectrometer. For an external standard, 99.9% D2O with 0.75% 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt was used. For 13C NMR spectroscopy, the sample was scanned for 4 days.

LC-MS and LC-MS/MS were performed with an in-line HPLC-TSQ 7000 mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with an electrospray (ESI) interface. The LC system consists of a quaternary pump P4000, an auto sampler AS3000, and a photo diode array detector UV6000LP. A Phenomenex LUNA 5µ C18 (2) 250- x 4.6-mm column was used for sample analysis. The column was eluted for 2 min with 25 mM formic acid-water (buffer A), at a flow rate of 0.6 ml per minute, and then with a linear gradient to 40% buffer B (100% acetonitrile in 25 mM formic acid) over 40 min, followed by a linear increase to 100% buffer B over the next 5 min. The eluant from the column was also monitored with an on-line photodiode array detector.

Data were processed with the software pack Xcalibur 1.2. The masses of the parent and daughter ions in the LC-MS and LC-MS/MS experiments described below were measured within a ±0.5-Thompsons (Th) window centered on the mass-to-charge ratio of interest. The collision energy used for MS/MS experiments was either 25 or 70 eV.

Quantitative Determination of K2P in Individual Normal Human Lenses and Brunescent Cataract Lenses—Thirty normal human lenses, ranging in age from 2 to 75 years, and 10 single brunescent cataract lenses (type I and II, classified based on the extent of browning of the lens) were decapsulated and homogenized individually in 0.5 ml of deionized water. The homogenate was centrifuged at 27,000 x g for 20 min, the supernatant was removed, and the pellet was washed twice with 0.5 ml of deionized water. The 27,000 x g pellet obtained was washed once again with 1 ml of deionized water and resuspended in 2.5 ml of deionized water. It was sonicated in a water bath for 6 min with a Heat Systems-Ultrasonic Inc. Sonicator (Model W-375) at a power setting of 4 and a duty cycle of 40%. The solubilized protein was recovered after centrifugation at 27,000 x g, representing at least 95% of the total WI fraction. The supernatants were combined and considered the WS fraction. The protein content of both WS and WI fractions was determined using a BCA assay kit (Pierce).

Both the WS and WI fractions were submitted individually to enzymatic digestion, as described above. The final digests of each single lens were applied to a small size-exclusion Bio-Gel P-2 chromatography (1 x 25 cm, bed volume 19.6 ml), with 25 mM formic acid as eluant. Fractions of 0.5 ml were collected and monitored for absorbance at 330 nm and for fluorescence at 450 nm when excited at 350 nm. Based on 330-nm readings, peak 1 was pooled and concentrated to 0.5 ml. Peak 1, 100 µl (20% of the total), was injected onto an analytical Prodigy HPLC column (4.6 x 250 mm, Phenomenex) for quantitative determination of K2P based on the absorbance at 343 nm. The peak area was converted to picomoles/mg of lens protein according to a standard calibration curve of purified K2P.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and Purification of K2P from Human Lens Protein Digests—A pooled cataract lens WISS protein digest (Fig. 1A) and a calf lens protein digest (panel B) were fractionated by Bio-Gel P-2 gel filtration chromatography (Fig. 1). Five peaks were pooled from the cataract lens WISS protein digest. Very few chromophores or fluorophores were detected from the calf lens protein digest, indicating that the cataract lens chromophores were not produced during the lengthy enzymatic digestion process or contributed by the added enzymes per se.



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  		FIG. 1.
Bio-Gel P-2 elution profile of a typical proteolytic digest of the water-insoluble sonicate supernatant (WISS) fraction from brunescent cataract (A) and calf lens protein control (B). The profiles for absorbance at 330- and 350/450-nm fluorescence are labeled in each panel. The numbers and arrows in panel A identify the peaks, which were pooled based upon 330-nm absorbance.

 
Peak 1 was concentrated and submitted to three additional purification steps by using preparative (Fig. 2A), semi-preparative (panel B), and analytical (panel C) reversed-phase HPLC (Fig. 2). The absorbance profiles at 280 and 343 nm are presented and labeled in each panel. The A343 peak marked by an asterisk is K2P. The final purified K2P recovery was 1.3 mg from 8–9 g of protein digest.



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  		FIG. 2.
Reversed-phase HPLC profiles showing the purification of K2P. Peak 1 was fractionated by preparative (A), semi-preparative (B), and analytical (C) Prodigy ODS columns. The absorbance values at 280 and 330 nm were monitored and labeled in each panel. The peak marked with an asterisk is K2P.

 
Structure Elucidation of K2P—The chemical structure of this novel chromophore was characterized by its absorption, fluorescence, 1H NMR, 13C NMR, COSY, HMQC, HMBC, NOESY, and TOCSY two-dimensional spectra and its mass spectroscopic properties. Its structure was determined to be 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium, to which we assigned the trivial name lysine-lysine pyridinium (K2P) (see Structure 1).



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  		STRUCTURE 1
 
Structure of K2P—The UV absorption spectra (Fig. 3) showed a maximum wavelength of K2P to be 343 nm at pH 7, which was unchanged at either pH 2 or 12. The emission maximum was 410 nm when excited at 343 nm (data not shown). The 343-nm absorbance is consistent with the structure of the conjugate and depends upon the presence of the nitrogens as part of the resonance structure.



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  		FIG. 3.
The UV absorption spectrum of K2P at pH 2, 7, and 12.

 
After purifications by preparative, semi-preparative, and analytical reversed-phase C18 columns, K2P was subjected to LC-MS and MS/MS analyses using water-acetonitrile solvents with formic acid as a counter ion (Fig. 4). As shown in panel A of Fig. 4, a single peak was eluted at 5.71 min and detected by its absorbance at 343 nm. The corresponding mass spectrum for this peak revealed a molecular ion peak at an m/z value of 370.96 Da (panels B and C), indicating a molecular mass of 369.96 Da. Based on the molecular mass, we examined a number of possible empirical formulas. Only C17H31N4O5 matched the rest of the analytical data as shown below. The LC-MS/MS fragmentation pattern of this compound is shown in panel D. The fragments, labeled as 326 (K2P-45 Da), 281 (K2P-45-45 Da), 196, 179, and 84, were assigned individually to the structure, as shown in panel E. The sequential loss of two 45-Da fragments suggests that two carboxyl groups may exist in the molecule. The lysine-derived fragments of 84 and 130 (not labeled) suggest that lysine is the possible parent amino acid for this compound.



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  		FIG. 4.
LC-MS and LC-MS/MS spectra of K2P. A, shows the HPLC profile monitored at 343 nm; B, shows the LC-MS at select m/z of 371 Da; C, the LC-MS spectrum of the peak that eluted between 5.72 to 6.46 min; D, the LC-MS/MS spectrum of K2P; and E, the suggested fragmentation pattern for K2P based on the data in panel D.

 
After K2P was purified by using formic acid as the counter ion, the sample was subjected to 1H and 13C NMR spectroscopy. A proton signal at a chemical shift ({delta}) of 8.1 ppm appeared in the 1H spectrum. This was likely due to residual formic acid, because when the sample was re-purified by analytical RP-HPLC with HFBA as the counter ion, the signal at 8.1 ppm disappeared. Fig. 5A shows 1H NMR spectrum of the final purified K2P in 100% D2O (full spectrum) or in H2O/D2O = 9/1 solution (see insets a and b).



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  		FIG. 5.
A, 1H NMR spectrum of K2P. 1H NMR spectrum was collected in D2O solvent. Insets a and b are resonances of H-6 and H-5 being collected in water/D2O (9/1) solvent. Insets c and d are the expansions of resonance of H-3 and H-2. Inset e shows the exchange of H-5 by deuterium; B, 13C NMR (panel b) and distortionless enhancement by polarization transfer (DEPT, panel a) spectra of K2P. Inset c is the expansions of resonance of {alpha}-carbonyl carbons of two lysines. The major signals are assigned and labeled in each panel.

 
The chemical shifts ({delta}, ppm) for each hydrogen atom, the number of protons, and the coupling constants (J, Hz) between the indicated protons are summarized in Table I. All the protons were assigned and labeled (Fig. 5A). Inset e shows the exchange of H-5 by deuterium (D) in D2O. When the sample was dissolved in 100% D2O, the doublet signal of H-5, which is coupled with H-6 at 5.24 ppm in H2O, disappeared in D2O (inset b), and the corresponding signal of H-6 (7.70 ppm) appeared as a singlet in D2O instead of its doublet signal in H2O (inset a). However, when we dried the sample and reconstituted the sample in H2O/D2O (v/v, 9/1) solvent, H-5 appeared again as a doublet and coupled with the H-6 (doublet) as shown in both insets a and b. As previously reported by Henle et al. (31) on an arginine-methylglyoxal condensed adduct, N{delta}-(5-methyl-4-oxo-5-hydroimidazol-2-yl)-L-ornithine, an exchange of the proton at C-5 in D2O was observed. The signals of H-3 (t, 4.60 ppm) and H-2, 2' (d, d, 3.63, and 3.73 ppm) were expanded in insets c and d, respectively. Furthermore, the integrated proton numbers and {alpha}, {beta}, {gamma}, {delta}, and {epsilon} proton signals from the two lysine residues were assigned separately. The downfield shift of methylene protons on the {epsilon}-carbon (3.58 ppm, 2H) suggests that the {epsilon}-nitrogen atom of lysine moiety is part of a heterocycle, which is in agreement with the structure suggested.


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  		TABLE I
1H and 13C NMR data of K2P (D2O/H2O, 25 °C)

 
Fig. 5B (panel a) shows the distortionless enhancement by polarization transfer (DEPT-135) spectrum, and panel b shows the 13C NMR. The sample used for 13C NMR was purified with formic acid as a counter ion, whereas the DEPT spectrum was gathered on a sample of K2P with HFBA as a counter ion. Therefore, a 13C signal of formic acid at 175 ppm was observed. Inset c shows the expanded signals for the two carboxyl-carbons of the lysine residues, which appear at 177.4 and 177.6 ppm, respectively. The chemical shifts ({delta}, ppm) for each carbon were summarized in Table I together with those of protons. DEPT is a spectral editing sequence. As shown in Fig. 5B (panel a), DEPT-135 give a spectrum with positive CH and CH3 signals and negative CH2 signals. Quaternary carbons are missing from the DEPT spectrum. By the comparison of 13C NMR and DEPT data, it is clear that C-2 was CH2, C-3, -5, and -6 were either CH or CH3 and C-4 was a quaternary carbon. Based on the integration result of 1H NMR, all of C-3, -5, and -6 can be identified as CH, but not CH3. Similarly, all carbon signals from the two lysine residues were assigned and labeled on the charts mentioned above.

The relationships between proton and proton, proton and carbons of H/C-2, -3, -4, -5, and -6 were considered. The H–H correlation spectroscopy (COSY) is shown in Fig. 6A, where H-3 is J-coupled to H-2 and H-2', and H-6 is J-coupled to H-5 even though the cross signal between H-6 and H-5 was relatively weak due to the slow exchange of H-5 by deuterium (refer to Fig. 5A).



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  		FIG. 6.
A, 1H–1H correlation spectroscopy (COSY) of K2P. The cross signals between H-3 and H-2, and between H-6 and H-5 are aligned in the panel; B, heteronuclear multiple quantum correlation (HMQC) spectroscopy of K2P; C, heteronuclear multiple bond correlation (HMBC) spectroscopy of K2P. The major cross signals between protons and carbons are aligned in the panel.

 
The heteronuclear multiple quantum correlation (HMQC) spectroscopy was used to determine which protons of the molecule are bonded to which 13C nuclei (or other X nuclei). As shown in Fig. 6B, the J-coupled correlation between H-2, H-2' and C-2, H-3 and C-3, and H-6 and C-6 can be assigned. In addition, all other cross signals once again could be assigned to two the lysine residues.

Based on the chemical shifts and the correlation between each proton and carbon, it can be concluded that position 2-CH2 J-coupled with position 3-CH; position 5-CH J-coupled with position 6-CH as a conjugate and the heterocycle ring between the two lysine residues consisted of {epsilon}-N, C-2, C-3, C-4, C-5, and C-6, as shown in the structure. However, it was not certain whether the {epsilon}-amino group connected to C-2 or C-3, {epsilon}'-amino group of another lysine residue and/or whether the hydroxyl group (-OH) connected to the C-3 or C-4 positions. Heteronuclear multiple bond correlation (HMBC) spectroscopy was used to clarify the exact position of the {epsilon} and {epsilon}'-amino group. HMBC is a modified version of HMQC and is suitable for determining the long range 1H-13C connectivity. By using this HMBC technique, the positions of C-2, C-3, C-4, C-5, C-6, and {epsilon}'-amino group were located. As shown in Fig. 6C, (a) cross-peaks between H-6 proton and C-2, C-4, C-5, and {epsilon}-carbon, (b) cross-peaks between H-2/2' protons and C-3, C-4, C-6, and {epsilon}-carbon, and (c) cross-peaks between {epsilon}-H protons and C-2, C-6, {delta}-carbon, and {gamma}-carbon were observed. These results indicated that the {epsilon}-amino group in the heterocycle ring was adjacent to C-2 and C-6. The crucial information for the connection of the {epsilon}'-amino group to C-4, but not to C-3, was obtained from the cross-peak between protons of {epsilon}'-carbon of lysine and C-4 (Fig. 6C). Therefore, we concluded that the {epsilon}'-amino group linked with C-4 and the hydroxyl group (-OH) linked with C-3. Considered together, the results of these structural analyses indicate that the structure of this novel yellow chromophore was 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium.

Quantification of K2P in Normal Human Lenses and Brunescent Cataract Lenses—Purified K2P was dried under vacuum to a constant weight, and 1.3 mg of K2P was finally recovered from 150 pooled aged normal human lenses and 350 pooled early stage brunescent cataract lenses. The acid stability of K2P was tested by analyzing the samples before and after acid hydrolysis. Although K2P was stable in acid solution (pH ~ 2) during our purification process, it was totally decomposed by acid hydrolysis with 6 M HCl at 110 °C for 20 h. Therefore, analyses of all the protein samples prepared from individual human lenses were carried out using our enzymatic digestion procedure.

The quantity of K2P in individual normal human lenses of different age and type I/II brunescent Indian cataract lenses, classified based on the extent of browning of the lens, was determined. The enzymatic digests of WS and WISS proteins from each individual lens were prepared. Peak 1 isolated from a small scale Bio-Gel P-2 column was analyzed by an analytical RP-HPLC with an online photodiode array detector. Typical HPLC profiles of peak 1 from individual cataract or aged normal human lens WISS digests are shown in Fig. 7. The peak marked by an asterisk is K2P, which was confirmed by both retention time and absorption spectrum. Twenty percent of the whole peak 1 by volume was subjected to HPLC, and the quantity of K2P in individual lens was calculated as picomoles per mg of lens protein, based on the peak area according to a standard curve.



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  		FIG. 7.
Analytical reversed-phase HPLC profiles for quantitative determination of K2P in individual lens protein digests. The samples from cataract-WISS, aged normal human lens-WISS, and calf lens protein (CLP) control are labeled in each profile. The peak marked with an asterisk is K2P.

 
The K2P levels in normal human lens WISS protein digests are shown in Fig. 8. Totally, 30 individual lenses of different age were analyzed. The results revealed an age-dependent increase in K2P, especially after 40 years. The highest samples from aged lenses reached ~400 pmol/mg of lens protein.



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  		FIG. 8.
Age-dependent increase of K2P in normal human lens WISS proteins.

 
Furthermore, 15 aged normal human lenses ranged from 62 to 75 years old (the average was 68 years old) and 10 type I or II brunescent cataract lenses, classified based on the extent of browning of the lens, were quantitatively analyzed for K2P in both WS and WISS protein digests. As shown in Fig. 9, the average levels of K2P in aged normal human lens WS and WISS protein digests were 22 ± 15 and 260 ± 93 pmol/mg of lens protein, respectively. In contrast, cataract lens WS and WISS protein digests contained 85 ± 51 and 613 ± 363 pmol/mg of lens protein, respectively. A large variation of K2P content was seen in the cataract WISS protein digests. In one particular case, the K2P level reached 1400 pmol/mg of lens protein.



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  		FIG. 9.
Quantitative determination of K2P in WS and WISS proteins from individual aged normal human and brunescent cataract lens.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
As the human lens ages, protein-bound yellow chromophores accumulate in the lens, particularly in the lens core. Senile cataracts are characterized by the accumulation of large protein aggregates that contain increased cross-linked crystallins and protein-bound browning products. Non-disulfide protein cross-links in lens tissue are increasingly seen in aging lenses and exist at higher levels during cataract formation. The identity of the major yellow chromophores bound to human lens proteins has been pursued by several investigators, but the origin of these protein-bound compounds and their effects on protein structure remain largely unknown.

Many of these browning compounds are proposed to be AGEs, which can absorb light in the UVA region (320–400 nm). Because UVA light that reaches the lens is 1000 times greater than UVB, the presence of these AGEs can amplify photodamage to lens proteins. As proteins become increasingly modified, they become part of the lens WI fraction. This fraction is composed of large protein aggregates that become insoluble upon homogenization. These proteins can be solubilized with urea initially, but as browning proceeds in brunescent cataracts, the aggregates can no longer be solubilized by both denaturing and reducing agents (10). These data argue for increased protein cross-linking that prevents dissociation.

Pentosidine is the classic AGE cross-link analyzed in aged tissues. Although pentosidine is present in tissues at very low levels, it serves as a useful bio-marker for AGE formation. Because pentosidine is acid-stable and highly fluorescent, it can be readily analyzed in protein hydrolates. Most AGEs, however, are acid-labile, and it is assumed that these acid-labile AGEs make a more quantitative contribution to protein cross-linking.

To search for significant acid-labile AGE cross-links, we used an enzymatic hydrolysis method to release the modified amino acids from lens proteins. Our enzymatic digestion procedure releases at least 95% free amino acids from both aged normal human lens proteins and cataract lens proteins (26–28). Treatment with Pronase alone, however, produced many incompletely digested peptides. To minimize oxidative degradation of modified amino acids, the digestion was carried out under argon and in the dark at all times. Yellow absorbance was monitored at 330 nm, because this wavelength exhibited the highest non-Trp absorbance present (32).

Bio-Gel P2 chromatography separated the yellow chromophores into five A330-absorbing peaks. A broad early-eluting peak, which may contain protein cross-links, represented 25 to 30% of the total A330 absorbance. The major peak in this fraction was identified and sufficient purified material allowed the isolation of 1.3 mg of K2P, which was sufficient for NMR spectral measurements.

LC-MS/MS (Fig. 4) and NMR spectroscopy (Figs. 5 and 6) unequivocally elucidated the structure of K2P as 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium, a cross-link between the {epsilon}-amino groups of two lysine residues. The formation pathway and biological functions of this cross-link in vivo are not known, but the presence of a pyridinium ring argues for glycation chemistry. This chemistry is currently under investigation. Structurally, K2P has two unusual features. One lysine {epsilon}-amino group is incorporated into the pyridinium ring, as seen with pentosidine, MOLD, GOLD, crossline, and vesperlysine A, however, the second lysine {epsilon}-amino group in K2P is attached to the ring. Also, the ring structure is not fully unsaturated. It is possible that the second lysine attaches to carbon 3 prior to ring closure, preventing the final ring dehydration step. It has been reported previously that conjugated Schiff bases (R1–NH–CH=CH–CH=N–R2), derived from amino acids and malondialdehyde, give a typical absorbance maximum at 370 and 435 nm (33). Therefore, the {lambda}max of 343 nm of K2P is consistent with the structure of the conjugate and depends upon the presence of the nitrogen as part of the resonance structure. Also, the formation of a completely unsaturated pyridinium ring would decrease the {lambda}max to ~280 nm (34).2

K2P level in individual human lens WS and WISS protein digests was quantitatively determined. The Bio-Gel P-2 size-exclusion fractionation technique and the specific absorbance of K2P at 343 nm allowed us to determine the K2P level directly from the peak 1 (Fig. 7). K2P levels in normal human lenses increased with age, especially after the age of 40 years (Fig. 8). The same pattern of human lens coloration (27) and the increases of other post-translational modifications of lens proteins, e.g. by UV filters (35–37) after middle age have been previously reported. A lens barrier hypothesis proposed by Truscott allows an explanation for these phenomena (38). Significantly higher levels of K2P were detected in cataract lenses and in water-insoluble proteins as compared with water-soluble proteins (Fig. 9) suggesting this cross-link may have a role in the formation of the water-insoluble proteins. The relatively high level of K2P in aged normal human lens reveals its potential importance in the aging and in the pathogenesis of cataracts.

For the past two decades, cross-links such as pentosidine, vesperlysine A, imidazolium cross-links (MOLD, GOLD), and glucosepan were detected in human lens proteins. Pentosidine and vesperlysine A, like K2P, exhibit UVA absorbance and increase with the age, but the levels of these AGEs are only 2 to 10 pmol/mg of lens proteins in aged normal human lenses, increasing to 20 pmol/mg of lens proteins in cataract lenses (21). K2P was present at 260 pmol/mg of lens protein in aged normal human lens, it increased to 610 pmol/mg of lens protein in brunescent cataract lens (Fig. 9). GOLD and MOLD increased with the age, too, and reached about 60 and 160 pmol/mg protein, respectively, in aged normal human lenses (23), but they had no absorbance in the UVA region. Therefore, as with yellow chromophores in human lens, K2P appears to be a major UVA sensitizer and protein cross-link identified to date. Indeed, K2P can be oxidized by UVA irradiation (data not shown). Determining the photochemistry of K2P will be one of our future objectives. The glucosepan level was reported to be 160–200 pmol/mg of lens protein in both aged normal human and brunescent cataract lenses (24). It seems a good marker for evaluating lens protein cross-linking.

Taken together, K2P may serve as a useful bio-marker for assessing the role of lens protein modification and coloration in aging and in the pathogenesis of cataract. There is no clear data now showing the formation pathway of K2P, even though we found an identical signal of m/z and retention time by LC-MS from an enzymatic digest of ascorbic acid modified calf lens proteins. To clarify this, the origin of K2P is being pursued.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant EY07070 and in part by a department grant from Research to Prevent Blindness, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Mason Eye Institute-East, The University of Missouri-Columbia, 404 Portland St., Columbia, MO 65201. Tel.: 573-882-6093; Fax: 573-884-4868; E-mail: chengr{at}health.missouri.edu.

1 The abbreviations used are: WI, water-insoluble; WISS, water-insoluble sonicate supernatant; WS, water-soluble; AGE, advanced glycation end-product; COSY, correlation spectroscopy; DEPT, distortionless enhancement by polarization transfer; HFBA, n-heptafluorobutyric acid; HMBC, heteronuclear multiple bond correlation; HMQC, heteronuclear multiple quantum correlation; LC-MS/MS, liquid chromatography-tandem mass spectrometry; ESI, electrospray ionization mass spectrometry; K2P, lysine-lysine-pyridinium or 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium; NOESY, nuclear Overhauser effect spectroscopy; RP-HPLC, reversed-phase high performance liquid chromatography; TOCSY, total correlation spectroscopy; Th, Thompsons (atomic mass unit); MOLD, methylglyoxal lysine dimer; GOLD, glyoxal lysine dimer. Back

2 R. Cheng, Q. Feng, and B. J. Ortwerth, unpublished data. Back


   ACKNOWLEDGMENTS
 
We are sincerely grateful to Dr. S. A. Patney for her tireless efforts in the collection of the cataract lenses used in this research. Special thanks to Wei Wycoff (NMR Facility, University of Missouri) for assistance with NMR spectroscopy.



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 RESULTS
 DISCUSSION
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