Figure 2. The amino acid sequences of the IRBP and variants.

REPEAT 1 REGION: The top sequence shows the amino acid sequence of wild type human IRBP as deduced from cDNA and genomic nucleotide sequences. Standard single letter codes are used to represent amino acids. The X represents an amino acid that could not be identified. The deduced sequence includes a signal peptide typical of secretory proteins at positions -22 to -6. This is followed by a propeptide, GPTHL, of five amino acids, from -5 to -1. In human IRBP derived from cadaver eyes about 50% of the protein begins with the glycine (G, position -5) of the propeptide (68). The remainder begins with phenylalanine (F, position +1 ). When wild type IRBP is first synthesized and secreted it appears that the protein is the full length form including the propeptide as shown here and starts at position -5. All the deletion variants begin at the same point, suggesting that they are similarly processed and secreted. The exception is R1, which still contains its signal peptide. How it avoids cotranslational processing that normally removes the signal peptide is unknown, but the mass of protein being produced may simply overwhelm the insect cell's signal peptidase. The E. coli expressed Repeat 1 protein, EcR1, yielded an amino acid sequence matching that expected from the DNA sequence of the clone, including a seven amino acid extension (underlined) before the beginning of Repeat 1. REPEAT 2 REGION: The E. coli expressed protein, EcR2, contains a short N-terminal extension of 17 amino acids (underlined) derived from the vector, followed by the IRBP repeat 2 sequence. The sequence shown here matches the sequence deduced from the DNA construct exactly. For the Repeat 3 and 4 regions, EcR3 and EcR4 have amino acid sequences identical to the expected seven amino acids encoded by the vector (underlined) followed exactly by the deduced sequences from each repeat. This figure shows that all the proteins encode IRBP by their identity with the deduced amino acid sequences from the cDNA and gene, and by similarity with amino acid sequences of IRBP from other species (68, 69).

REPEAT 1 REGION:

-22              -5   +1
|                |    |
MMREWVLLMSVLLCGLAGPTHLFQPSV...  SEQUENCE DEDUCED FROM THE GENE AND cDNA
 MREXVLLM                       SEQUENCE OF R1
                 GPTH           SEQUENCE OF WT
                 GPTXLFQP       SEQUENCE OF R12-
                 GPTHLFQP       SEQUENCE OF R12+ UPPER BAND
                 GPTHLFQPSV...  SEQUENCE OF R12+ MIDDLE BAND
                 GPTHLFQPSV...  SEQUENCE OF R12+ LOWER BAND
                 GPTHL....      SEQUENCE OF R123 (Predominant)
                      FQPSV...  SEQUENCE OF R123 (Minor)
                                SEQUENCE OF G719S
                                SEQUENCE OF R725C
          MVPSSDPGPTHLFQ        SEQUENCE OF EcR1

REPEAT 2 REGION:

                 RSALPGV...     SEQUENCE DEDUCED FROM THE GENE AND cDNA
MVPSSDPLVTAASVLEFRSALPGV        SEQUENCE OF EcR2

REPEAT 3 REGION:

       QSL...                   SEQUENCE DEDUCED FROM THE GENE and cDNA
MVPSSDPQSL                      SEQUENCE OF EcR3

REPEAT 4 REGION:

       AKVPT...                 SEQUENCE DEDUCED FROM THE GENE AND cDNA
MVPSSDPAKVPT                    SEQUENCE OF EcR4