Table I. Identification of crystallins in fetal bovine lens shown in Figure 2A.

The identities of the crystallins labeled in Figure 2A were determined by partial Edman sequence analysis of blotted proteins from 2-DE gels. The identities of ß-crystallins in red were determined by direct Edman sequencing of the truncated and resulting unblocked N-termini. Numbers in parenthesis indicate the number of residues missing from the N-terminus, based on reported sequences in Swiss-Prot and Genbank databases. Numbering begins with the N-terminal methionine as residue 1, even though this residue may be absent in the parent protein. Intact [gamma]-crystallins were also directly sequenced because they are normally unblocked. The remaining N-terminally blocked [alpha]- and ß-crystallins were identified by sequencing of internal tryptic fragments. The ßB3 marked with an asterisk was of lower molecular weight than unmodified ßB3, but unlike the other shortened ß-crystallins contained a blocked N-termini. Regions marked [gamma]D/E in Figure 2A may contain either [gamma]D or [gamma]E, since the N-terminal sequences determined matched the reported sequences of both proteins.

An "X" in the "Sequence Determined" indicates where an amino acid identity could not be determined.

The partial sequences determined by Edman degradation matched the given residue numbers of the previously reported sequences of bovine crystallins appearing in the given Swiss-Prot or Genbank entry. The numbering of residues again included the N-terminal methionine as residue 1. However, this residue may be missing in the actual protein. Note that the given partial sequence for ßA4 does not match the numbers of the sequence of ßA4 appearing in P11842, since recent sequences of chicken ßA4 (P49152) and human ßA4 (P53673), and the measured mass of bovine ßA4 [24] indicated that the actual sequence is 14 residues shorter at the N-terminus than reported in P11842.

Crystallin   Sequence Determined   Residues   Accession Number

ßB1          AELPPGSYK              53-61     P07318
ßB1 (-15)    PGPDGKGKAGPPP          16-28     P07318
ßB3          GEQYVLEK               76-83     2338456
ßB2          DSGDFGAPQP            173-182    P02522
ßB3*         MEIVDDDVPSL           129-139    2338456
ßA3 (-11)    SLPTTKMAQT             12-21     P11843
ßA3 (-22)    PMPGSVGPWKIXIY         23-36     P11843
ßA1          EWGSHAQTSQ            180-189    P11843
ßA2          NYSEFGTQAH            176-185    P26444
ßA3          GYQYILEXDH            178-187    P11843
ßA4          IVVWDEEGFQGR           14-25     P11842
[alpha]B1    PFFPFHSPS              13-21     P02510
[alpha]B2    HFSPEELK               83-90     P02510
[alpha]A1    VQEDFVEIHG             89-98     P02470
[alpha]A2    TVLDSGISEV             55-64     P02470
[gamma]S     PVDWGAASP             160-168    P06504
[gamma]D/E   GKITFYEDRGFQGRH         2-16     P08209/P23005
[gamma]D/E   GKITFYEDRGFQGRH         2-16     P08209/P23005

Table II. Identification of crystallins in adult bovine lens shown in Figure 2B.

The identities of the crystallins labeled in Figure 2B were determined by partial Edman sequence analysis of blotted proteins from 2-DE gels. The identities of ßB2 (-8), ßB3 (-22), and both acidic and basic forms of ßA3 (-22) were determined by direct Edman sequencing of the truncated and resulting unblocked N-termini (red). The identity of [gamma]S was determined by Edman sequencing of an internal tryptic fragment (black). Numbers in parenthesis give the number of residues missing from the N-terminus of the truncated proteins based on the previously reported sequences of each crystallin in the given Swiss-Prot or Genbank entry.

All partial sequences matched the reported sequences in Swiss-Prot or Genbank entries, except residue 11 of ßB2 which yielded alanine (in green) instead of the reported lysine at this position in P02522. This alanine was likely an artifact, since residue 11 of a m-calpain cleaved bovine ßB2 was confirmed as lysine in Table III.

The identities of other labeled crystallin subunits in Figure 2B were determined by comparison of their migration positions to crystallins identified in fetal lenses shown in Figure 2Aand Table I.

Crystallin           Sequence Determined   Residues   Accession Number

ßB2 (-8)             AGAPQPLNPK              9-18     P02522
ßB3 (-22)            SYKVIVYEME             23-32     2338456
ßA3 (-22) (basic)    PMPGSVGPWK             23-32     P11843
ßA3 (-22) (acidic)   PMPGSVGPWK             23-32     P11843
[gamma]S             VLEGAWIFYE            132-141    P06504

Table III. Partially truncated ß-crystallins observed following incubation of fetal bovine lens soluble protein with m-calpain

Soluble protein from fetal bovine lenses was incubated for 10 min at a ratio of 5 units m-calpain/mg lens protein, the mixture separated by 2-DE, and five distinct regions containing partially truncated ß-crystallins with free N-termini either produced by m-calpain or found prior to m-calpain incubation subjected to Edman sequence analysis. The analyzed regions correspond to areas circled in green on the 2-DE gel shown in the third panel of Figure 3.

The partially truncated ß-crystallin(s) identified in each region which either increased in concentration or first appeared following m-calpain incubation are shown in green. Underlined ß-crystallins produced by m-calpain in vitro were also observed in vivo in either fetal or adult bovine lenses (see Table I and Table II). Unlike the other ß-crystallins listed, ßA3 (-22) shown in red did not increase following incubation, but is shown here since it co-migrated with the m-calpain digestion product ßA3 (-17). Numbers in parenthesis give the number of residues missing from the N-terminus of the truncated proteins based on the previously reported sequences of each crystallin in the given Swiss-Prot or Genbank entries.

Three of the five regions contained mixtures of two proteins which did not fully resolve during 2-DE. However, the presence of only two amino acids per cycle allowed the identification of the two truncated ß-crystallins present.

The experimentally determined sequences in the second column matched the listed residue numbers of the previously known sequences of bovine ß-crystallins in the given Swiss-Prot or Genbank entries.

Crystallin   Sequence determined   Residues   Accession Number

ßB1 (-12)    AVNPGPDGKG            13-22      P07318
ßB1 (-15)    PGPDGKGKAG            16-25      P07318

ßA3 (-11)    SLPTTKMAQTNPMPG       12-26      P11843

ßB2 (-8)     AGKPQPLNPKIIIFE        9-23      P02522
ßB3 (-10)    AAAGKSHGGLGGSYK       11-25      2338456

ßB3 (-5)     STPEQAAA               6-13      2338456

ßA3 (-17)    MAQTNPMP              18-25      P11843
ßA3 (-22)    PMPGSVGP              23-30      P11843