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(Investigative Ophthalmology and Visual Science. 2000;41:175-182.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Characterization of Human Lens Major Intrinsic Protein Structure

Kevin L. Schey1, Mark Little1, John G. Fowler1 and Rosalie K. Crouch2

From the Departments of 1 Cell and Molecular Pharmacology and 2 Ophthalmology, Medical University of South Carolina, Charleston.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the primary covalent structure of human lens major intrinsic protein (MIP) in lenses of varying age.

METHODS. MIP was isolated from single human lenses of various ages (7–86 years) by homogenization of the lenses, followed by centrifugation and urea washes of the membranes. Proteins present in the membrane preparation were reduced, alkylated, and cleaved by CNBr. Peptide fragments were fractionated by reverse-phase high-performance liquid chromatography, and the primary structures of the peptides were determined by tandem mass spectrometry and Edman sequencing.

RESULTS. Complete coverage of the human MIP sequence was observed in the form of CNBr fragments. In addition, peptide structures resulting from in vivo heterogeneous N- and C-terminal cleavage were characterized. The amount of intact MIP decreased with lens age; however, the pattern of truncation did not change from 7 to 86 years. The major site of phosphorylation was identified as serine 235. Asparagine residues 246 and 259 were completely deamidated by age 7 years.

CONCLUSIONS. The major structural modifications of human lens MIP have been determined. Human MIP is heterogeneously modified in lenses ranging in age from 7 to 86 years of age by N- and C-terminal truncation, phosphorylation, and deamidation, resulting in decreased levels of native intact MIP with age.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The major intrinsic protein (MIP) of the lens is the most abundant lens membrane protein, comprising approximately 50% of the lens membrane protein content. As a member of the aquaporin family of proteins, it is believed that MIP functions as a water channel in the fiber cell membranes to regulate water and small molecule movement.1 A variety of studies have shown that MIP, in reconstituted membrane preparations2 or expressed in Xenopus oocytes,3 4 5 can facilitate ion, water, and glycerol transport. In addition, one study has demonstrated a role for MIP in cell-to-cell adhesion in the lens.6 MIP function is clearly important in maintaining lens transparency as evidenced by the discovery of two mutations in the MIP gene which cause altered C-terminal MIP sequences to be expressed, resulting in cataracts in mice.7 Several additional lines of evidence indicate the importance of the C-terminal region of MIP, including a report that phosphorylation of MIP altered voltage-dependence properties on ion transport8 and reports of calmodulin binding to the C-terminal domain of MIP9 10 and trypsin inhibition of calmodulin-induced channel closure.11

The amino acid sequence of bovine MIP was deduced from the cDNA, and a model has been proposed that consists of six transmembrane spanning {alpha}-helices.12 The human MIP sequence was subsequently determined, revealing 92% identity and 98% homology to the bovine sequence.13 Studies on the MIP structure suggest that MIPs exist in tetrameric form in lens membranes.14 Cryo-electron microscopy studies of the erythrocyte aquaporin (AQP1) at 6 to 7 Å resolution indicate that members of the aquaporin family do indeed contain six transmembrane {alpha}-helical domains and exist as tetramers in the membrane.15 16 A recent electron microscopy study on lens MIP at 9 Å resolution showed a structure similar to that of AQP1.17 However, specific positions of amino acids in the membrane and modifications to those amino acids cannot be determined at the resolution of electron microscopy. In addition, the hydrophobic nature of MIP precludes x-ray crystallographic analysis and makes primary structure analysis challenging. Hence, alternative methods are needed to obtain detailed structure information. In early structural studies, cyanogen bromide (CNBr) cleavage was used to generate two main fragments,18 19 one of which was partially sequenced by Edman degradation. In our studies, we have likewise cleaved the protein with CNBr and used mass spectrometry (MS) to analyze all the fragments from bovine and rat MIPs.20 21 We have identified the predominant phosphorylation site as Ser 235 in bovine and rat MIP, a conserved residue among all known MIP sequences, and specific N- and C-terminal truncation products in MIP isolated from rat selenite cataract lenses.

Changes in MIP structure during lens maturation and in cataract models have been examined in a variety of studies; however, to date no specific structures have been reported for human MIP. The major human MIP maturation product, MIP22, was first identified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE).22 23 This product, observed in lenses as young as 2 years of age, increased with lens age and increased in nuclear fractions relative to cortical fractions. Subsequent examination of age-related changes in human lens MIP revealed N-terminal and C-terminal modifications by reduction in binding of antibodies specific for N- and C-terminal sequences.24 Truncation was believed to be the primary cause of loss of antibody recognition because of the concomitant increase in MIP22 observed by SDS–PAGE. The only detailed structural information on MIP modification was determined for bovine MIP, where N-terminal truncation was identified at residue 17,25 and C-terminal deamidation was discovered at Asn 246.26 Examination of MIP structure in animal cataract models revealed similar MIP modifications/truncation products.27 28 In a recent study of MIP isolated from selenite-induced cataracts in rats, specific sites of truncation were identified that implicated calpain, although not exclusively, as a primary candidate for proteolytic truncation of MIP.21 Although detailed structural data have been reported for MIP modifications, the fact remains that specific sites and structures of modifications to human lens MIP remain undetermined.

We applied our approach to membrane protein structure elucidation to determine the primary structure of human lens MIP. We identified the primary sites of phosphorylation, as well as deamidation, and discovered a specific pattern of truncation common to lenses of varying age. These results provide a framework from which to interpret age and cataractous changes in membrane protein structure in human lenses.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Protein Isolation
Human lenses were obtained from donations to the South Carolina Lions Eye Bank. All experiments were carried out according to the tenets of the Declaration of Helsinki. Fourteen lenses, ranging in age from 7 to 86 years, were decapsulated and homogenized by hand in 1 mM NaHCO3 buffer (pH 8.0), 5 mM EDTA, and 10 mM NaF. The homogenate was centrifuged (135,000g), and the resultant pellet was washed with 5 mM Tris (pH 9.0), 1 mM EDTA, and 1 mM CaCl2, then washed with Tris buffer containing 4 M urea, and finally washed with Tris containing 7 M urea. One preparation was carried out in the presence of a broad-spectrum protease inhibitor cocktail (Boehringer–Mannheim, Indianapolis, IN) to prevent the possibility of proteolysis during preparation.

Protein Cleavage
The enriched MIP membranes were suspended in 1:1 n-propanol/1.5 M Tris buffer at pH 8.7, and a 1000 molar excess of tributyl phosphine over cysteine content and a 2400 M excess of 4-vinylpyridine were added. The reaction was terminated by a water wash. To delipidate the protein, the membranes were suspended in 95% ethanol overnight at -20°C and centrifuged at 135,000g. The pellet was then washed with acetone followed by water. MIP was solubilized in 75% trifluoroacetic acid (TFA), and a 500 molar excess of cyanogen bromide (CNBr) over methionine content was added. The reaction was carried out under argon in the dark at room temperature for 18 hours. The digest was terminated by a fivefold dilution with water and speed vacuum-dried.

Protein Analysis
The dried CNBr digest was solubilized in 5 µl TFA, 42 µl acetonitrile, 84 µl isopropanol, and 4.875 ml water. Peptides were fractionated over a 2.1 x 100 mm Aquapore C4 column with a gradient of 97.5% H2O, 0.05% TFA to 97.5% isopropanol/acetonitrile (2/1), 0.05% TFA over 112 minutes at a flow of 400 µl/min. A post-column split (1:9) was used to direct 10% of the flow into the mass spectrometer, and 90% of the eluant was collected for off-line mass spectrometric analysis or secondary digestion with trypsin.

High-performance liquid chromatography (HPLC)–purified C-terminal peptides were solubilized in (NH4)HCO3 buffer (pH 8.5) with 10% acetonitrile. Trypsin was added in a 1:10 enzyme/peptide ratio, and the digests carried out for 16 to 22 hours at 37°C. The lyophilized tryptic digests were solubilized in 0.1 M acetic acid, 10% acetonitrile, and separated on a Microtech (Saratoga, CA) 320-µm inner diameter C4 capillary column with a gradient of 95% 0.1 M acetic acid to 97% acetonitrile over 60 minutes at a flow rate of 10 µl/min. Some tryptic mixtures were subjected to alkylation by adding methanolic HCl (5 µl), prepared by drop-wise addition of 150 µl of acetyl chloride to 1 ml ice-cold methanol while stirring, to dried peptide for 30 minutes and the reaction was quenched by speed vacuum drying. Tryptic peptides were also subjected to Edman sequencing (PE Biosystems Procise 494 sequencer, Foster City, CA) after separation on a capillary C18 HPLC column.

Mass Spectrometry
Two mass spectrometric approaches were taken to characterize the separated MIP CNBr products eluting from the HPLC. First, fractions were collected and off-line mass spectrometric analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry was carried out in a PE Biosystems Voyager-DE mass spectrometer (Framingham, MA). Second, the HPLC effluent was directed into a Finnigan (San Jose, CA) LCQ electrospray (ESI) tandem mass spectrometer for online MS and tandem MS (MS/MS) analysis. For MALDI analysis peptides were solubilized in either 85% acetic acid or 70% acetonitrile/0.1% TFA and mixed (1:3 µl) with the matrix {alpha}-cyano-4-hydroxycinnamic acid in 70% acetonitrile, 0.1% TFA. The peptide/matrix solutions were placed on the sample plate to dry. Typically, 256 laser shots were averaged to produce the mass spectra, and expected mass accuracy is 0.02% for external calibration with peptide standards from PE Biosystems. Laser power was optimized for signal intensity and resolution and varied between 1400 and 1700 on the Voyager-DE instrument.

Online HPLC ESI-MS and tandem mass spectrometry (MS/MS) experiments were carried out in automated fashion. Briefly, as the HPLC effluent entered the LCQ instrument, a mass spectrum was acquired and the most abundant ion was isolated for subsequent structural elucidation. The isolated ion of interest was fragmented via collisions with helium background gas to generate sequence specific fragmentation. The fragment ion masses were recorded by a second scan of the instrument, providing an MS/MS spectrum. This process was continuously repeated throughout the entire HPLC run, resulting in sequence information on each abundant peptide that eluted from the HPLC. LCQ software was used to calculate peak areas for ion signals of eluting CNBr peptides.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
In an effort to examine structural changes in human MIP, single human lenses varying in age from 7 to 86 years of age were used to prepare MIP-enriched membrane fractions. Given the hydrophobicity of this integral membrane protein, chemical cleavage using CNBr was combined with reverse-phase HPLC to fractionate the protein into fragments suited to mass spectrometric analysis. Representative chromatograms are shown in Figure 1 for 17- and 75-year-old lenses. Labeled peaks corresponding to MIP sequences were assigned on the basis of measured molecular masses by MALDI mass spectrometry (Table 1) . Note that the intensities of the N- and C-terminal peptides in HPLC chromatograms decreased with the age of the lens relative to the internal sequences 47 to 81 and 82 to 90. This general trend was observed with age both by HPLC analysis (data not shown) and by mass spectrometric analysis (discussed below). The mass spectral data described in Table 1 demonstrate that the entire MIP sequence, corresponding to the published translated sequence,13 is observed as isolated CNBr fragments thereby allowing subsequent detailed structural analysis of MIP products. The small peptide corresponding to residues 177 to 183 (molecular weight [MW] 713.7 Da) elutes very early in the chromatogram and is observed in online LC-MS experiments. Other peptides isolated by HPLC have been sequenced and identified as fragments of different lens crystallins and MP20, which are isolated as part of the membrane fraction and are the subjects of ongoing investigations.



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  		Figure 1. HPLC chromatograms of human MIP CNBr cleavage products from 17-year-old (A) and 75-year-old (B) lenses using slightly different gradient times.

 

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  		Table 1. Summary of Observed MIP26 CNBr Fragments

 
Phosphorylation
The mass spectrum of the C-terminal peptide (184–263) has a signal 80 Da higher than the predicted molecular weight indicative of phosphorylation. The phosphopeptide signal intensity is typically 25% to 40% that of the unmodified C-terminal peptide, and no significant change in intensity was observed with age. To identify the site of phosphorylation, the isolated C-terminal CNBr peptide was cleaved with trypsin and the products subjected to LC-MS/MS analysis. Strong signals were observed at mass-to-charge (m/z) ratios of 606.5 and 1211.6 (Fig. 2A ), which corresponded to the singly, [M + H]+, and doubly, [M + 2H]2+, charged molecular ions of the phosphorylated peptide 229–238 (calculated MW 1211.3 Da). Tandem mass spectrometry was used to verify the peptide sequence and identify the phosphorylation site. The peptide of interest, m/z 606.5, was mass selected and fragmented. The fragment ions were mass analyzed to produce an MS/MS spectrum. Because peptides fragment predictably along the peptide backbone at amide bonds,29 and because phosphorylated peptides readily lose phosphoric acid from fragment and molecular ions,30 MS/MS data were interpreted to give information on peptide sequence and modification. MS/MS sequence analysis (Fig. 2B) of the putative phosphopeptide 229–238 provided unambiguous evidence that the phosphorylation site is serine 235. No other phosphorylated MIP peptides were observed. The major fragment ion at m/z 557 corresponds to loss of phosphoric acid, the [M + 2H–H3PO4]2+ ion, from the doubly charged precursor ion at m/z 606.5. Sequence-specific fragments indicate that the N-terminal 6 residues (b6 ion at m/z 687) and the C-terminal 2 residues (y2 ion at m/z 260) are unmodified, thereby eliminating serines 229 and 231 as possible sites of phosphorylation. Fragment ions containing serine 235 appear shifted by 80 Da indicative of a phosphoserine residue and/or appear after loss of phosphoric acid (asterisks) providing proof of the phosphorylation site.



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  		Figure 2. (A) Electrospray mass spectrum of the putative tryptic phosphopeptide 229–238 from a 15-year-old lens, indicating the [M + H]+ ion at m/z 1211.6 and the doubly charged [M + 2H]2+ ion at m/z 606.5 and (B) MS/MS spectrum of phosphorylated peptide 229–238 [M + 2H]2+ ion at m/z 606.5. Sequence ions are labeled according to Biemann’s nomenclature,29 and asterisks indicate loss of phosphoric acid.

 
Deamidation
Because C-terminal deamidation was reported in bovine MIP,26 we examined the C-terminus of human MIP for deamidation. Deamidation of asparagine and glutamine residues to aspartic acid and glutamic acid residues causes a 1 Da increase in the peptide molecular weight. Due to difficulty in chromatographic and mass spectrometric resolution of these closely related products, putative deamidated C-terminal tryptic peptides were alkylated at carboxylic acid sites by exposure to methanolic HCl, followed by tandem mass spectrometric sequencing. Unalkylated peptides were HPLC separated and subjected to Edman sequencing. The peptide 239–263 and truncation product 239–259, predominant peptides observed in C-terminal tryptic digests, each contain four acidic amino acids and a C-terminal carboxylic acid group, all of which are expected to be esterified. The MALDI mass spectrum of the tryptic peptide mixture from the C-terminus of a 27-year-old lens (Fig. 3A ) indicates that 7 methyl groups were added to the tryptic peptides 239–259 and 239–263 as evidenced by an increase in molecular weight of 98 Da to each at m/z 2234.8 (predicted 2236.3) and 2651.9 (predicted 2649.8), respectively. The two most likely sites of additional alkylation sites are deamidated Asn 246 and Asn 259, because glutamine residues (248 and 261) are less likely to be deamidated.31 Note that there is little heterogeneity in the MALDI molecular weights for these two peptides, indicating that these peptides are completely deamidated. The other signals in the spectrum correspond to other tryptic products and are shifted by the expected molecular weights according to the acidic groups present in their sequence. The presence of these signals rules out artifactual deamidation/alkylation, because each contains two asparagine residues that are not alkylated. The MS/MS spectrum of the alkylated 239–59 from a 7-year-old lens is shown in Figure 3B . The peaks marked by asterisks provide conclusive evidence that Asn 246 and Asn 259 are present as aspartic acid residues. Edman sequencing of HPLC-purified 239–263 confirmed this result. In addition, as residue 246 eluted from the sequencer, the protein signal dropped by roughly 67%, suggestive of non-enzymatic deamidation, which leads to an abundance of ß-isoaspartic acid, a blocked residue to Edman sequencing. In all experiments, there was very little evidence of native nondeamidated peptides. In a 62-year-old lens there was some evidence of an eighth alkyl group addition; however, the exact site of this addition was not determined.



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  		Figure 3. (A) MALDI mass spectrum of a tryptic digest of HPLC-purified MIP C-terminal CNBr peptide 184–263 from a 27-year-old lens after alkylation and (B) MS/MS spectrum of the alkylated 239–259 peptide. Superscripts indicate the number of methyl (M) groups attached to the peptide. Asterisks indicate critical signals that establish deamidation sites at Asn 246 and Asn 259.

 
Truncation
Significant signals were observed in mass spectra of human MIP CNBr peptides, which elute near the intact C-terminal peptide and which can be assigned to truncated sequences by comparing measured molecular weights with calculated molecular weights of truncated CNBr fragments. The MALDI mass spectrum of C-terminal CNBr products from an 81-year-old lens is shown in Figure 4 , indicating a series of signals corresponding to truncated peptides. Truncation appears to occur between residues 228 and 259, corresponding to the predicted C-terminal tail of the MIP structure.12 MALDI mass spectra of C-terminus–containing fractions from lenses varying in age from 7 to 86 years are shown in Figure 5 . Remarkably, the pattern of truncation is nearly identical in lenses ranging in age over 7 decades, suggesting that the pattern of MIP truncation is established early in life. What does change with age is the intensity of the intact C-terminus signal (MW 8638), which decreases with age. Integration of UV and mass spectrometry signals provides complementary information indicating a loss of intact MIP with age. Quantitative analysis of MIP truncation is presented below. A MIP preparation was carried out on a 27-year-old pair of lenses from a single donor in the presence and absence of a broad-spectrum protease inhibitor cocktail to rule out the possibility of proteolytic cleavage during MIP preparation. Observed truncation patterns (data not shown) were identical with each other and to the data in Figure 5 . The observed variation in signal intensity for the 47–90 peptide at m/z 4656 is likely due to fraction collection times, because this peptide elutes immediately before the C-terminal peptide 184–263.



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  		Figure 4. MALDI mass spectrum of an HPLC fraction eluting at 57–59' containing MIP C-terminal truncated peptides from an 81-year-old human lens.

 


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  		Figure 5. MALDI mass spectra of MIP C-terminal fractions from human lenses varying in age from 7 to 86 years.

 
The MALDI mass spectra of N-terminal CNBr products from a 62-year-old lens are shown in Figure 6 , indicating truncation after residues 1, 2, 3, 5, 6, and 7. The large signals marked by asterisks in the spectra at m/z 4908, 5033, and 5219 correspond to carbamylated N-terminal peptides 4–46 (predicted m/z 4904), 3–46 (5033), and 2–46 (5220), respectively. This is a result of washing lens membranes with old urea contaminated with isocyanic acid, which reacts with lysines and N-terminal amino groups.32 The fact that the truncated peptides 2–, 3–, 4–46 (and to a lesser extent 6–, 7–, and 8–46) are carbamylated at their N-termini (no lysines are present in the MIP N-terminal sequence) in the first membrane wash establishes that truncation at these sites occurs in vivo, before any tissue manipulations with the exception of homogenization. MALDI mass spectra of N-terminus–containing fractions from lenses varying in age from 7 to 81 years are shown in Figure 7 . As was observed for C-terminal truncation, the truncation patterns are nearly identical over 7 decades; however, the signal is degraded by the age of 81 years. Although it is difficult to see in the MALDI data, the signals for the intact N-terminus peptide decrease with age and this effect was quantitated by LC-MS experiments.



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  		Figure 6. MALDI mass spectrum of an HPLC fraction eluting at 81–83' containing MIP N-terminal truncated peptides from a 62-year-old human lens.

 


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  		Figure 7. MALDI mass spectra of MIP N-terminal fractions from human lenses varying in age from 7 to 81 years.

 
The MALDI data provide a qualitative view of peptide modification in human MIP26; however, this tool is not adequate for quantitative determinations of the extent of modification. Therefore, electrospray mass spectrometric signals of HPLC-separated MIP CNBr products were used to quantitate the degree of protein truncation by comparing the signal intensities for terminal peptides to internal sequences. The results, plotted in Figure 8 , indicate an age-dependent linear decrease in N- and C-terminal signal intensities relative to the internal sequence 47–81.



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  		Figure 8. Plot of N-terminal ({blacksquare}) and C-terminal ({circ}) ion signals compared with the signal for the internal sequence 47–81 as a function of lens age. Signals were obtained from ion intensity plots from LC-MS experiments and are summed ion intensities for ions of all charge states related to the sequences 1–46 and 2–46 (N-terminal), 184–263 (C-terminal), and 47–81 (internal).

 
Molecular weight searches were carried out in LC-MS data sets for all possible truncated peptides. Many of the signals observed in the MALDI data were of low abundance or absent in the electrospray LC-MS data and were therefore not quantifiable. However, this search revealed several truncated products that elute at times different from the intact N- and C-terminal peptides, including sequences 30–46, 36–46, 38–46, 184–196, and 184–211. Interestingly, truncation occurring in these regions, according to the predicted model,12 would indicate extracellular cleavage. All truncated peptide signals are summarized in Table 2 .


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  		Table 2. Summary of Observed MIP Truncation Products

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
For the first time, the complete structure of human MIP has been mapped using a combination of chemical and proteolytic cleavage, reverse-phase HPLC, and mass spectrometry. Figure 9 shows a diagram of the MIP molecule, indicating approximate transmembrane domains and sites of modification. Investigation of the intact protein revealed that the predominant site of phosphorylation is serine 235 and that this site is typically found to be modified at the 25% to 40% level in all lenses studied. Identification of phosphoserine 235 is consistent with our previous findings in bovine20 and rat21 MIPs. It is inconsistent with the first reported site, serine 243, in bovine MIP,33 and, more importantly, residue 243 exists as an aspartic acid residue in the human MIP sequence. The operative kinase and the functional role of MIP phosphorylation remain to be elucidated.



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  		Figure 9. Diagram of MIP sequence indicating approximate transmembrane domains (boxes), sites of CNBr cleavage (methionine residues bolded), and sites of modification.

 
Examination of proteolytic fragments of the MIP C-terminus revealed two deamidation sites: one at Asn 246 is consistent with the site found in bovine lenses and a second new site at Asn 259. Perhaps it is not surprising to find deamidation in MIP given the growing literature on deamidation of lens crystallins.34 35 36 What is surprising is the finding of complete deamidation of C-terminal asparagine residues 246 and 259 by the age of 7 years. The susceptibility of asparagine deamidation based on the preceding and succeeding residues suggests that residues 246 and 259 are the most susceptible of all C-terminal asparagine residues to deamidation.31 The operative mechanism of deamidation in MIP appears to be a nonenzymatic reaction given the blocked peptide at residue 246, likely due to ß-isoaspartic acid generated in the deamidation reaction. The functional consequences of creating two negative charges in the MIP C-terminus, a putative regulatory region, remain unknown.

A detailed investigation of human MIP structure revealed major N- and C-terminal truncation products in lenses as young as 7 years of age. A heterogeneous truncation pattern is observed with major sites identified as residues 2–8, 29, 35, 37, 196, 211, 239, 243–248, and 259. Examination of MIP truncation with age indicates that the same N- and C-terminal sites are cleaved in MIP over at least 7 decades. Both the HPLC and mass spectrometric data reveal that the amount of intact MIP decreases with age, which is in agreement with the earlier SDS–PAGE22 23 and antibody recognition24 studies.

The heterogeneous nature of the truncation products observed in this study suggest either a nonspecific cleavage mechanism or a multiplicity of mechanisms occurring in the lens. Major cleavage sites correspond to those observed in the selenite cataract model in rats,21 which were interpreted as mainly, but not solely, due to calpain. Invoking intracellular calpain would not explain the apparent cleavage at putative extramembranous portions of the protein. Extracellular cleavage may result from extracellular proteases, like the recently described lenticular extracellular metalloproteinases,37 or may be due to membrane damage that causes exposure of extracellular domains to intracellular proteases. Another possibility is that a more general nonspecific oxidative cleavage is occurring. Interestingly, a cleavage mechanism has been proposed that involves asparagine residues of lens {alpha}-crystallin, which could also account for truncation products observed at residues 246 and 259.38 Of equal importance to those observed truncation sites are those sites that are protected from cleavage. Because the truncation is at nearly every residue, the data suggest where intramembranous domains begin and end. For example, residues 9 to 29 are protected from cleavage and may represent intramembranous domain one, whereas residues 196 to 211 may represent intramembranous domain six. Extensive data searches were done to determine other intramembranous fragments; however, none was found.

Interpretation of the age-related truncation results is complicated by the fact that the entire lens was homogenized and analyzed. It may be that, as has been reported previously,22 23 the nuclear MIP is deteriorated to a greater extent than cortical MIP but that the presence of younger cortical MIP in our preparation mutes these changes. An alternative possibility is that the same cleavage processes occur throughout the lens regardless of fiber cell age. The approach used in this study is currently being used to examine human MIP structure in dissected cortical and nuclear fractions.

The functional consequences of N- and C-terminal truncation remain unclear. Given the putative regulatory role of the MIP C-terminus as implied by C-terminal phosphorylation and calmodulin binding, as well as phosphorylation effects on ion transport,8 the likelihood of C-terminal truncation altering MIP function seems high.

In summary, the covalent structure of human lens MIP has been examined in detail by an approach combining chemical cleavage, reverse-phase HPLC, and mass spectrometry. The predominant sites of phosphorylation, deamidation, and numerous sites of N- and C-terminal truncation are reported. The amount of intact MIP decreases with age; however, the pattern of truncation is similar with lens age and is established by 7 years of age. The functional significance of these results remains to be elucidated.


   Acknowledgements
 
Technical support was provided by MUSC Mass Spectrometry and Biotechnology Facilities.


   Footnotes
 
Supported by Grant EY-10722 from National Institutes of Health (Bethesda, Maryland; KLS) and Research to Prevent Blindness (New York, New York).

Submitted for publication March 17, 1999; revised August 8, 1999; accepted August 27 1999.

Commercial relationships policy: N.

Corresponding author: Kevin L. Schey, Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425. scheykl{at}musc.edu


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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