The Role of alpha - and epsilon -Amino Groups in the Glycation-mediated Cross-linking of gamma B-crystallin
STUDY OF THREE SITE-DIRECTED MUTANTS*

(Received for publication, February 14, 1997, and in revised form, March 31, 1997)

Hui-Ren Zhao Dagger , Ramanakoppa H. Nagaraj § and Edathara C. Abraham Dagger

From the Dagger  Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912 and § Center for Vision Research, Department of Ophthalmology, Case Western Reserve University, Cleveland, Ohio 44106

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

In the previous report we demonstrated that gamma B-crystallin is glycated predominantly at the N-terminal alpha -amino group (Casey, E. B., Zhao, H. R., and Abraham, E. C. (1995) J. Biol. Chem. 270, 20781-20786). To investigate the possible role of alpha - and epsilon -amino groups of gamma B-crystallin in glycation-mediated cross-linking, Lys-2 or Lys-163, or both, were mutated to threonine by site-directed mutagenesis in bovine gamma B-crystallin cDNA. Wild type and mutant gamma B-crystallins were expressed in Escherichia coli cells. Cross-linking studies were performed by incubating wild type and mutant gamma B-crystallins with glyceraldehyde, ribose, and galactose followed by SDS-polyacrylamide gel electrophoresis under reducing conditions. When both of the lysines of gamma B-crystallin were mutated to threonines (gamma B-K2T/K163T), the quantity of cross-linked products was greatly reduced, indicating that, despite the fact that the alpha -amino group is a major glycated site, epsilon -amino groups play a predominant role in cross-linking. Therefore, cross-linking ability depends not only upon the level of glycation but also upon which amino group is glycated. Steric hindrance may decrease the cross-linking ability of the alpha -amino group. Our results also show that Lys-2 and Lys-163 play almost equal roles in cross-linking of gamma B-crystallin. By incubating carbonic anhydrase, a protein with a blocked N terminus, and our novel "no lysine" gamma B (gamma B-K2T/K163T) with sugar, we were able to show for the first time that significant cross-linking occurs between lysines and non-lysine sites. The fact that pentosidine and imidazolysine, formed from ribose and methylglyoxal, respectively, were present in the cross-linked gamma B-crystallins revealed the existence of Lys-Arg and Lys-Lys cross-linking.


INTRODUCTION

Aldehyde or keto groups of reducing sugars react mainly with alpha - and epsilon -amino groups on proteins forming unstable Schiff base adducts, which rearrange to form more stable Amadori products (1). These Amadori products undergo a series of chemical reactions, becoming brown, fluorescent, and irreversibly cross-linked products. Vlassara et al. (2) first used the term advanced glycosylation (glycation) end products (AGEs)1 to describe these cross-linked products formed during the late stage of the Maillard reaction in vivo. AGE-mediated cross-linking is believed to be important in the pathogenesis of aging and in the complications of diabetes (3-5). Cross-linking of proteins is believed to occur mostly among epsilon -amino groups of lysines as well as guanidinium groups of arginines. But the roles of these groups and of alpha -amino groups in glycation-mediated cross-linking are still not clear because AGE structure is largely unknown. At present, AGEs are thought to be composed of diverse structures. However, studies with recently developed monoclonal and polyclonal antibodies against AGEs have suggested that they all have a common structure (6, 7). Elucidated AGE structures include Nepsilon -(carboxymethyl)lysine (8), pentosidine (9), pyrraline (10), Nepsilon -(carboxymethyl)hydroxylysine (11), crossline (12), and imidazolysine (13, 14). Among them, pentosidine (Lys-Arg), crosslines (Lys-Lys), and imidazolysine (Lys-Lys) have cross-linking properties.

gamma -Crystallin is one of the major classes of lens-soluble proteins. Bovine gamma B-crystallin, the major species of bovine gamma -crystallin, contains only two lysines (Lys-2 and Lys-163) among its 174 amino acid residues (15). gamma -Crystallin is the only crystallin with a free N terminus (16). It is synthesized at an early stage in the development of the lens and is found mainly at the core region of the mammalian lens (17). Since there is little or no protein turnover in the core region (18), postsynthetic modifications of gamma B-crystallin such as glycation will accumulate in the nucleus of the lens. Indeed, it is the core region where nuclear cataract, the important form of cataract with protein cross-linking and brunescense in humans, is formed.

In a previous report, we showed that the N-terminal alpha -amino group of bovine gamma B-crystallin is the predominant glycation site (19), as confirmed recently by mass spectrometry analysis (20). In the present communication, the two lysines of bovine gamma B-crystallin were mutated to threonine; this "no lysine" gamma B-crystallin, as well as the mutants containing only Lys-2 or Lys-163, was used to study the role of the N-terminal alpha -amino group of Gly-1 and the epsilon -amino groups of two lysines in glycation-mediated cross-linking of gamma B-crystallin.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis

The bovine gamma B-crystallin cDNA in pBluescript (15) was kindly provided by Dr. Regine Hay (Department of Ophthalmology, Washington University). gamma B-crystallin cDNA was subcloned into the NcoI/HindIII cloning site of expression vector pMON5743 (pMONgamma B), and site-directed mutagenesis of Lys-2 to Thr (pMONgamma B-K2T) was carried out using polymerase chain reaction as described previously (19). Creation of two new mutants, Lys-163 to Thr (K163T) as well as Lys-2 and Lys-163 to Thr (K2T/K163T), was accomplished with synthetic oligonucleotides. The desired base changes of Lys-163 to Thr were incorporated into the oligonucleotides (AACCAACTGTGGCATTC and AATGCCACAGTTGGTTC), and mutagenesis was performed by two-stage polymerase chain reaction-based overlap extension (21). Using pMONgamma B as a template led to the generation of a gamma B-K163T mutant, whereas when pMONgamma B-K2T was used as the template, the double mutant gamma B-K2T/K163T was generated. The nucleotide sequences were checked using the sequencing kit from U. S. Biochemical Corp. Other methods used in subcloning were described previously (19).

Expression and Purification of Recombinant gamma B-crystallins

Clones containing pMONgamma B, pMONgamma B-K2T, pMONgamma B-K163T, and pMONgamma B-K2T/K163T were used to express recombinant gamma B-crystallins of wild type, mutant K2T (gamma B-K2T), mutant K163T (gamma B-K163T), and mutant K2T/K163T (gamma B-K2T/K163T), respectively. Expression of wild type and mutant gamma B-crystallins was performed according to the method of Olins and Rangwala (22). Wild type and mutant gamma B-crystallins in the cell lysates were purified using cation-exchange HPLC according to the method of Siezen et al. (23) with minor modifications (19). Since gamma B-K2T/K163T has a lower pI, buffers used in cation-exchange HPLC were adjusted to pH 5.5 instead of pH 6.0, and bacterial proteins found in the gamma B-K2T/K163T fraction were removed by molecular sieve chromatography on a 2 × 100-cm Sephadex G-100 (Sigma) column.

SDS-PAGE and Western Blot

SDS-PAGE was performed according to the method of Laemmli (24) using a 12% separating gel and a 4% stacking gel under reducing conditions. Gels were routinely stained with Coomassie Blue, but in the case of galactose incubation a silver stain kit (Bio-Rad) was used to visualize small amounts of cross-linked products. Western blotting was carried out using a monoclonal antibody against rat gamma -crystallin (19, 25). Bovine gamma -crystallin was prepared as described previously (19).

Cross-linking of the Recombinant gamma B-crystallins with Sugars

Fifty microliters of wild type or mutant gamma B-crystallin (2 mg/ml) were incubated with or without sugar in darkness at 37 °C in phosphate-buffered saline (pH 7.4) containing 0.02% sodium azide. Since the cross-linking ability of different sugars varies (26), different sugar concentrations were used: glyceraldehyde (5 mM), ribose (100 mM), and galactose (1 M) (all from Sigma). To investigate the ability of lysine to cross-link with non-lysine sites (e.g. alpha -amino group as well as arginine and histidine residues), equal concentrations of carbonic anhydrase (CA) and gamma B-K2T/K163T or wild type gamma B were incubated with 5 mM glyceraldehyde. After incubation for 10 days (glyceraldehyde and ribose) or 20 days (galactose), the samples were analyzed by SDS-PAGE under reducing conditions, and the gels were scanned on a CS-9301 PC dual wavelength Fly Spot scanning densitometer (Shimadzu Corp., Tokyo, Japan). Protein concentration was determined by the method of Lowry et al. (27).

Estimation of Pentosidine and Imidazolysine by Reversed-phase HPLC

Pentosidine (Lys-Arg cross-link) was synthesized according to the method of Sell and Monnier (9). Imidazolysine (Lys-Lys cross-link) was synthesized as described previously (14). Wild type and mutant gamma B-crystallin (2 mg/ml) were incubated with either 25 mM D-ribose (for pentosidine) or methylglyoxal (for imidazolysine) in phosphate-buffered saline at 37 °C. Aliquots of 0.3 ml were withdrawn on days 0, 5, and 10. Proteins incubated simultaneously without the added carbohydrate served as controls. After incubation, the protein was hydrolyzed with 6 N HCl at 110 °C for 20 h. The acid was evaporated in a Speed Vac concentrator (Savant Instruments, Inc., Farmingdale, NY), and the amino acids were reconstituted in water followed by centrifugation at 14,000 × g for clarification. Pentosidine was estimated by reversed-phase HPLC on a C18 column (Vydac, 218TP, 25 × 0.46 cm, 10 µm, Separations Group, Hesperia, CA). The mobile phase consisted of water (A) and 60% acetonitrile in water (B) with 0.01 M heptafluorobutyric acid (Sigma). The gradient program used was 16-28% B in 35 min. The effluent from the column was monitored for fluorescence at 385 nm (excitation, 335 nm). Pentosidine eluted at ~29.0 min. For quantification, the peak area in the samples was compared with the peak area of a known amount of purified pentosidine. Other details of the HPLC settings are described elsewhere (28). Imidazolysine was estimated by reversed-phase HPLC on a C18 column (Vydac, 201HS54, 25 × 0.46 cm, 5 µm). The mobile phase consisted of water (A) and 70% methanol in water (B) with 0.1% heptafluorobutyric acid. The gradient program was 0-100% B in 35 min. The column effluent was allowed to mix with o-phthalaldehyde for postcolumn derivatization, and the fluorescent derivatives were detected by an on-line fluorescence detector (Jasco International Co. Ltd., Toyko, Japan) set at excitation/emission of 340/455 nm as described by Nagaraj et al. (14). Under these conditions imidazolysine eluted at ~22 min. For quantification, the peak area in the samples was compared with the peak area of a known amount of purified imidazolysine.


RESULTS

Expression, Purification, and Characterization of Recombinant gamma B-crystallins

Wild type and mutant gamma B-crystallins were expressed in JM101 Escherichia coli cells. The supernatants of cell lysates were concentrated and analyzed on 12% SDS-PAGE. Heavy bands with molecular mass equal to calf gamma -crystallin were found in all cell lysates except in the lysate of the cells containing the vector pMON5743 without gamma B-crystallin cDNA (Fig. 1A). The recombinant proteins were purified, and the purity of the proteins was checked on SDS-PAGE (Fig. 1B). Western blotting with a monoclonal antibody to rat gamma -crystallin (19, 25) confirmed the expression of gamma B-crystallin (Fig. 1B).


Fig. 1. Expression and characterization of wild type and mutant gamma B-crystallins. A, SDS-PAGE of protein extracts from cell lysates. Proteins were run on a 12% gel under reducing conditions and then stained with Coomassie Blue. All the cell lysates contained large amounts of ~20-kDa proteins comigrating with total calf gamma -crystallin except for the lysate of the cells that contained the vector pMON5743 without gamma B-crystallin cDNA (control). STD, molecular mass standards. B, SDS-PAGE and Western blot of purified recombinant gamma B-crystallins and total calf gamma -crystallin. Wild type and mutant gamma B-crystallins were purified by cation-exchange and molecular sieve chromatography, and the purity was checked on SDS-PAGE (right). Proteins were transferred to a polyvinylidene difluoride membrane and then probed with a monoclonal antibody to rat gamma -crystallin. The bands were visualized by alkaline phosphatase stain (left).
[View Larger Version of this Image (68K GIF file)]

Glycation-mediated Cross-linking

To cross-link gamma B-crystallins in a realistic in vitro time span, purified wild type and mutant gamma B-crystallins were reacted with different concentrations of sugars that are known to glycate and cross-link much faster than glucose and subjected to SDS-PAGE. After a 10-day incubation with DL-glyceraldehyde, dimer (~40 kDa), trimer (~60 kDa), and higher molecular mass cross-linked products were seen in wild type gamma B (Fig. 2A). Fewer cross-linked products were found in gamma B-K2T and gamma B-K163T, but heavy dimer bands could still be seen in both the mutants. If gamma B-K2T and gamma B-K163T are compared, the former seems to have slightly less cross-linked protein than the latter. Only a faint band of cross-linked product was observed with the double mutant gamma B-K2T/K163T despite the fact that the alpha -amino group of Gly-1 is the predominant glycation site of gamma B-crystallin. Gel scanning showed that formation of the dimer of gamma B-K2T/K163T decreased by 73% as compared with that of wild type gamma B. When other slower reacting sugars, such as ribose and galactose, were incubated with wild type and mutant gamma B-crystallins, similar results were obtained (Fig. 2, B and C), but no significant difference in cross-linking was seen among the wild type gamma B, gamma B-K2T, and gamma B-K163T, and no trimer or higher cross-linking product was observed. Again, only faint cross-linked dimer bands were found in the incubation of gamma B-K2T/K163T, which confirmed that epsilon -amino groups of lysine are important in glycation-mediated cross-linking.


Fig. 2. Cross-linking of wild type and mutant gamma B-crystallins by glyceraldehyde (A), ribose (B), and galactose (C). Recombinant proteins (2 mg/ml) were incubated with 5 mM glyceraldehyde and 0.1 M ribose for 10 days and 1 M galactose for 20 days. After incubation, proteins were run on a 12% gel under reducing conditions. Coomassie Blue stain (glyceraldehyde and ribose) or silver stain (galactose) was used to visualize the cross-linking products. STD, molecular mass standards.
[View Larger Version of this Image (39K GIF file)]

To investigate possible cross-linking between epsilon -amino groups and non-lysine cross-linking sites (e.g. alpha -amino group as well as arginine and histidine residues), CA was incubated with gamma B-K2T/K163T or wild type gamma B in the presence of 5 mM glyceraldehyde for 10 days. CA was chosen because it has a blocked N terminus, 18 lysines (29), and a molecular mass of about 29 kDa, which facilitates distinguishing the different cross-linked dimers, e.g. gamma B-K2T/K163T-gamma B-K2T/K163T, gamma B-K2T/K163T-CA, and CA-CA, on SDS-PAGE. Fig. 3 shows that there are substantial cross-linked products consisting of gamma B-K2T/K163T and CA (lane 2), and the quantity of the cross-linked product is much higher than that made up of two gamma B-K2T/K163T mutants (lane 5) but less than that comprised of wild type gamma B-crystallin and CA (lane 3).


Fig. 3. Cross-linking of carbonic anhydrase with recombinant gamma B-crystallins. CA, a lysine provider, was incubated with gamma B-K2T/K163T, wild type gamma B-crystallin, or itself in the presence of 5 mM glyceraldehyde for 10 days. The protein concentration was 2 mg/ml. Lane 1, molecular mass standards; lane 2, gamma B-K2T/K163T + CA; lane 3, wild type gamma B + CA; lane 4, CA only; lane 5, gamma B-K2T/K163T only.
[View Larger Version of this Image (102K GIF file)]

Formation of Pentosidine and Imidazolysine

To confirm the existence of Lys-Arg and Lys-Lys cross-linking, pentosidine and imidazolysine were analyzed after cross-linking recombinant gamma B with sugars (Fig. 4). Our data show that pentosidine and imidazolysine do exist in the cross-linking of gamma B-crystallin and the amounts of pentosidine and imidazolysine decrease along with the decrease of lysine content of gamma B. Fig. 4 also shows that approximately the same amount of pentosidine or imidazolysine was found from gamma B-K2T and K163T. This finding agrees with our cross-linking data, which show that gamma B-K2T and gamma B-K163T incubations contain about the same quantities of cross-linking products (Fig. 2A). Some pentosidine and imidazolysine detected in gamma B-K2T/K163T could have come from a small amount of bacterial protein contamination. Since gamma B-crystallin is a low lysine content protein, small amounts of high lysine content bacterial proteins may cause significant increases of pentosidine and imidazolysine even though the cross-linking of bacterial proteins cannot be seen on the SDS-PAGE of the protein cross-linking (Fig. 2).


Fig. 4. Estimation of pentosidine and imidazolysine. Formation of Maillard protein-cross-link pentosidine in proteins incubated with D-ribose (A) and protein-cross-link imidazolysine in proteins incubated with methylglyoxal (B) are shown. Proteins were incubated with or without 25 mM carbohydrate. The protein was acid-hydrolyzed for the estimation of protein cross-links by HPLC. See "Experimental Procedures" for details on HPLC procedures. In pentosidine assay, 1 unit of protein is ~1 mg of protein, and in imidazolysine assay, 1 unit of protein is ~225 µg of protein. The open symbols represent proteins that were incubated with the carbohydrate, and the closed symbol (black-square) represents proteins that were incubated without the carbohydrate (controls). open circle , wild type gamma B-crystallins; down-triangle, gamma B-K2T; square , gamma B-K163T; triangle , gamma B-K2T/K163T.
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DISCUSSION

Determination of the roles that specific glycated amino acid residues play in cross-linking and insolubilization of crystallins will shed light on the mechanism by which diabetic and senile cataracts form. From our results, it is clear that lysine, although only weakly glycated, is important for glycation-mediated cross-linking, and the alpha -amino group of gamma B-crystallin, despite being a major glycation site (19, 20, 30), has much less ability to cross-link with itself or with other residues. Therefore, cross-linking ability depends not only upon the level of glycation but also upon which amino group is glycated. Furthermore, by using our novel "no lysine" gamma B-crystallin (gamma B-K2T/K163T) and another protein having no free N terminus, we were able to show for the first time that significant cross-linking between lysine and non-lysine cross-linking sites exists (Fig. 3). Noticeable loss of arginine and lysine after incubating lysozyme with glucose suggests that lysine and arginine are important candidates for cross-linking (31). Earlier studies (9) have shown that lysine and arginine can be cross-linked by a pentose to form a Lys-Arg cross-link, namely pentosidine. Pentose could be formed from hexose through sugar fragmentation or from triose, tetrose, and ketose by condensation and/or reverse aldose reactions (32). Recently, Nagaraj et al. (14) showed that a three-carbon metabolite, methylglyoxal, which increases in diabetic lenses, reacts with lysines of proteins forming a Lys-Lys cross-link, namely imidazolysine. Thus, pentosidine and imidazolysine were analyzed in cross-linked gamma B-crystallin. The fact that pentosidine and imidazolysine are present in the cross-linking of gamma B-crystallin reveals the existence of Lys-Arg and Lys-Lys cross-linking. The high levels of imidazolysine (5-9 nmol/23 nmol of lysine) may have resulted from the possible lysine-rich protein contamination on crystallin preparation. Experiments with ultrapure preparations of proteins may shed light on this issue.

According to Prabhakaram and Ortwerth (33), [14C]lysine was more readily incorporated into lysozyme than [14C]leucine during glycation-mediated cross-linking studies. This finding was interpreted as evidence for cross-linking principally at the epsilon -amino group rather than at the alpha -amino group. This hypothesis was confirmed by our protein-protein cross-linking model. Okitani et al. (31) show that acetylation of free amino groups of lysozyme before incubation with glucose prevents cross-linking of the protein and loss of lysine and arginine. This finding strengthens the importance of the amino group in cross-linking. By using site-directed mutagenesis, we were able to differentiate between the roles of alpha - and epsilon -amino groups and show that the epsilon -amino group is more important than the alpha -amino group in cross-linking. The present study shows for the first time that an engineered "no lysine" protein is useful in assessing the role of alpha - and epsilon -amino groups in glycation-mediated cross-linking. The advantage of site-directed mutagenesis over the chemical modification is that the roles of individual amino groups of proteins in cross-linking can be ascertained.

The reason the readily glycated alpha -amino group has a weaker ability to cross-link with itself or other non-lysine sites than lysine is not clear. The N-terminal alpha -amino group is, in fact, on the surface of gamma B-crystallin (34) and is thus accessible to other cross-linking sites. One possible explanation is that cross-linking at this site is greatly decreased by steric hindrance. Interestingly, an earlier study by Beswick and Harding (35) shows that changes in the conformation of gamma -crystallin take place after glycation with glucose 6-phosphate, and this may prevent cross-linking at the alpha -amino group. Prabhakaram and Ortwerth (33) observe that incorporation of [14C]leucine into protein is ~20% that obtained with lysine. We also noted that although alpha -amino groups have much less cross-linking ability than epsilon -amino groups, cross-linking studies with gamma B-K2T/K163T suggest that the alpha -amino group of Gly-1 could form some low level cross-links with itself or with arginine and histidine residues (Fig. 2).

By incubating CA, used as an epsilon -amino group provider, with gamma B-K2T/K163T, we were able to show that there was significant cross-linking between the epsilon -amino groups of CA and the non-lysine cross-linking sites of gamma B-K2T/K163T (Fig. 3). We noted that the quantity of cross-linking products found in the incubation containing CA and wild type gamma B (lane 3) is higher than that found in CA and gamma B-K2T/K163T incubation (lane 2). This increased quantity could come from the cross-linking of lysine to lysine and/or lysines of wild type gamma B to non-lysine sites of CA. When CA alone is incubated with glyceraldehyde, a clear dimer band can be observed on SDS-PAGE, but when CA is incubated with wild type gamma B or gamma B-K2T/K163T, the CA-CA dimer bands almost disappear (Fig. 3). CA seems to cross-link more readily with gamma B-crystallin than with itself, and thus the major cross-linking product is CA-gamma B rather than CA-CA.

Arginines and histidines are thought to be involved in glycation-mediated cross-linking (9, 31, 36). Even though bovine gamma B-crystallin contains 16 arginines and 5 histidines, when both lysines were mutated to threonine, cross-linking was greatly reduced, suggesting that they act only as cross-linking "acceptors" of the glycated epsilon -amino group.


FOOTNOTES

*   This work was supported by Research Grants EY07394, EY11352 (to E. C. A.), and EY09912 (to R. H. N.) from the National Eye Institute and an unrestricted grant support from Research to Prevent Blindness to the Department of Ophthalmology, Case Western Reserve University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical College of Georgia, 1120 15th St., Augusta, GA 30912-2100. Tel.: 706-721-9526; Fax: 706-721-6300.
1   The abbreviations used are: AGE, advanced glycation end product; CA, carbonic anhydrase; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.

ACKNOWLEDGEMENTS

We express our appreciation to Dr. Regine Hay for the gift of bovine gamma B-crystallin cDNA and to Monsanto Company for the gift of pMON5743 expression vector.


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