The cDNAs and their derived amino acid sequences described above are highly homologous to human CD33. The similarity is highest a t the 5' end in the extracellular Ig domains, where 66 % of the nucleotides and 61
% of the amino acids are identical. Fig. 4.10 depicts the alignm ent between the two amino acid sequences. As expected for Ig-like proteins all of the extracellular cysteine residues are conserved between the murine and human proteins. The N-linked glycosylation sites are also maintained, while the possible GAG (Ser-Gly-Ala-Gly) attachm ent site in the m urine sequence is not present in hum an CD33. However, another type of putative GAG attachm ent site, Ser-Gly, is found in the hum an CD33 sequence in a sim ilar position, near to the transm em brane domain (residue 252). The homology between the hum an and m urine CD33 sequences is interrupted ju st before the transm em brane domain (the dashed lines in Fig. 4.10), in the region containing the putative GPI signal sequence in m urine CD33 (RKS, position 235). A putative GPI anchor signal sequence is also found in the hum an sequences (DGS, position 248).
Alignment of the transmembrane regions reveals a 55% amino-acid identity. The homology w ith the hum an sequence extends w ithin the cytoplasmic region of the shorter murine m33-A isoform, although a t a
* * ** ** *★ * * * ***** ***** * * * _ * * ^ * * * * _ * * h u m a n C D 3 3 LLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNS PVHGYWFREG 64 m 3 3 - A LPLFLLCAGSLAQDLEFQLVAPESVTVEEGLÇVHVPÇSVFYPSIKLTL-GPVTGSWLRKG 63 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * AIISGDSPVATNKLDQEVQEETQGRFRLLGDPSRNI^SLSIVDARRRDNGSYFFRMERGS 124 VSLHEDSPVATSDPRQLVQKATQGRFQLLGDPQKHDÇSLFIRDAQKNDTGMYFFRWREP 123 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * -TKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTÇSVSWAÇEQGTPPIFSWLSAA 183 FVRYSYKKSQLSLHVTSLSRTPDIIIPGTLEAGYPSNLTÇSVPWAÇEQGTPPTFSWMSTA 183 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * PTSLGPRTTHSSVLIITPRPQDHGTNLTÇQVKFAGAGVTTERTIQLNVTYVPQNPTTGIF 243 LTSLSSRTTDSSVLTFTPQPQDHGTKLTÇLVTFSGAGVTVERTIQLNVT RK--- 234 * * * * * * * _ * * * * * * * * * * * * _ * * * . . . . * * PGDGSGKQETRAGLVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGSNDTHPTT 3 03 SG-Q-MRE-LYLYM£EMYK^LLIiiSLC^ ZLIMFCRRKTTK--L-S--VHMGC 281 * * * * * * * * * * * * * GSASPKHQKNSKLHGPTETSSCSGAAPTVEMDEELHYA-SLNFHGMNP 3 50 ENPIKAHQQDSKVHSNPENPRPLQKDSPQE-QSSVHTKISLDFMGGKP 32 8 F ig u re 4.10: C o m p a risio n of th e h u m a n a n d m u rin e CD33 p ro te in sequences
The hum an CD33 sequence is shown above the m urine m33-A sequence. The single amino acid letter code is used. Identity is represented by asterisks and conservative substitutions by dots. Gaps introduced in the sequence to maximize the alignment are designated by dashed lines. The conserved cysteine residues are underlined. The putative transmembrane domain is double underlined. The sequences were aligned with Bio Search Software.
much lower level (29% amino acid identity). Significant divergence, however, occurs between the 3’ cytoplasmic domain of the hum an CD33 and murine m33-B isoform. The multiple phosphorylation sites identified in both murine isoforms are not conserved in the hum an CD33 sequence. In the hum an form one CK2 phosphorylation site is identified at position 358 and two serine/threonine PKC consensus phosphorylation sequence at positions 288 and 311 are predicted.
The overall amino acid identity between the hum an and murine mCD33-A isoform is 53% and considering the conservative replacements the similarity reaches 71%. Comparisons of murine CD33 to other proteins in the databases are discussed in the next chapter.
4.3. D iscu ssio n
In this chapter the molecular cloning of the murine homologue of the hum an CD33 myeloid antigen is described. The experimental design employed to isolate this was based on the assumptions, th a t they are likely to be homologous and th a t the expression of murine CD33, like th a t of hum an, should be restricted principally to haemopoietic cells of myelomonocytic lineage. The cloning strategy used included PCR analysis and screening of a mouse bone marrow cDNA library, from which two cDNA clones differing by an 83 bp insertion were isolated.
PCR amplification has found extensive application in molecular cloning and it has been a commonly used technique for the generation of specific probes for uncloned genes (Lee et al., 1988; Girgis et al., 1988; Buck and Axel, 1991; Torres et al., 1992). The poor PCR results, with both degenerate and specific hum an CD33 prim ers, described here were probably due to the inability to select the right prim er combinations and/or too stringent PCR conditions. The degenerate and specific hum an CD33 oligonucleotide prim ers, used in th is study, were random ly
designed, matching sequences within the beginning and the end of the hum an CD33 cDNA, with the expectation of amplifying larger fragments of the mouse gene. Some of the primers, such as D l, D2, H2, H3 and H6, lie in reasonably conserved regions as was shown later by the comparison between the hum an and m urine CD33 sequences. The lack of PCR products in the case of degenerate primers, D l and D2 (in both D l and D2 pools there were primers showing more than 90% identity a t the nucleic acid level to the murine sequence), was probably due to insufficiently good homology of th e anti-sense D3 prim er to the m urine sequence. D3 sequence corresponds to a poorly conserved region in the cytoplasmic domain, where only one of the six amino acids is identical between the hum an and murine sequence (residues 313-318 of m33-A and 338-344 of hum an CD33, with the degenerate nucleotide primers having less th an 30% homology to the murine sequence).
The absence of specific mouse PCR products w ith the relatively homologous H2 (60% identity on nucleic acid level to the respective m urine sequence), H3 (75% identity) and H6 (77% identity) prim ers, probably can be attrib u ted to too stringent PCR conditions - 57°C annealing tem perature. The PCR amplification and cloning of the hum an CD33 fragm ent also confirmed the high sensitivity of PCR and the extreme precautions needed against contamination. Such precautions are essential when there are concentrated solutions of target DNA nearby (the cDNA from CD33 positive human KGl cells).
On the other han d PCR very much sim plified th e cloning procedures. For example amplification of inserts in the bacteriophage X
vectors using oligonucleotide primers annealing to the flanking vector sequences or the isolation of the 5' end of the murine gene by 5'RACE. However, a limitation of the current method is its relatively high rate of m isincorporation. The Taq polymerase lacks editing functions and
incorporates an incorrect nucleotide at a rate of 2 xlQ-4 nucleotides per cycle in polymerase chain reactions. This ra te of m isincorporation tra n s la te s into an overall error frequency of 0.25% in 30 cycle amplification (Saiki et al., 1988), making the sequence of an individual DNA molecule cloned from an amplified pool unreliable. To eliminate the possibility of misincorporations, the H3/H6 and p33-RACE sequences were confirmed by sequencing three additional independent PCR clones, while for p33-A and p33-B, because of th e ir size, conventional cloning procedures were preferred instead of PCR.
The cDNAs and derived amino acid sequences reported here show extensive similarity to hum an CD33 (71% overall amino acid similarity). The two cDNA clones encode two mouse CD33 isoforms, m33-A and m33- B, w ith distinct cytoplasmic regions of 67 and 136 aa, respectively, in contrast to a single CD33 form identified in hum an (Simmons and Seed,
1988). The regions with the most significant sim ilarities between the hum an and mouse species include the first and second Ig-like domains. Less conserved are the cytoplasmic domains, with the most significant divergence in their C-termini occurring, between the hum an CD33 and m urine m33-B isoform. Multiple phosphorylation sites were found in the cytoplasmic regions of murine CD33 isoforms, suggesting th a t they may be phosphoproteins, b ut w hether they have a role in in tracellu lar signalling rem ains to be determ ined. Additional insights into the biological function of m urine CD33 homologue and its distinctive cytoplasmic domains may be provided by further sequence analysis, which is discussed in the next chapter.
Chapter 5
SEQUENCE COMPARISONS OF MURINE